MECHANISMS OF β- LACTAMASE INHIBITION AND
HETEROTROPIC ALLOSTERIC REGULATION OF
AN ENGINEERED β- LACTAMASE-MBP FUSION PROTEIN
By
WEI KE
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy.
Dissertation Advisor: Dr. Focco ven den Akker
Department of Biochemistry
CASE WESTERN RESERVE UNIVERSITY
May, 2011
CASE WESTERN RESERVE UNVERISTY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Wei Ke .
candidate for the Ph.D degree*.
(signed)Paul Carey . (chair of the committee)
Focco van den Akker .
Menachem Shoham .
Robert A. Bonomo .
Marion Skalweit .
______
(date) 23 March, 2011
*We also certify that written approval has been obtained for any proprietary material contained therein.
TABLE OF CONTENTS
LIST OF TABLES ………………………………………………………………………8
LIST OF FIGURES ………………………………...…………………………………9
ACKNOWLEDGEMENTS ...………………………………………………………..13
LIST OF ABBREVIATIONS …………………...……………………………………15
ABSTRACT ……………………………….………………………………………...17
CHAPTER 1 Background and Significance …………………………………………....18
1.1 Antibiotic Resistance Crisis……………….…………………………….…...18
1.2 β-lactamases overview……………………………………………………….19
1.3 β-Lactam antibiotics and β-lactamase inhibitors ……………………………23
1.4 Structures of class A β –lactamases …………………………………………25
CHAPTER 2 Crystal Structures of SHV-1 β-Lactamase in Complex with Boronic Acid Transition State Inhibitors …………………………………………………...…….32
2.1 Introduction ………………………………………………………………….32
2.2 Materials and Methods ………………………………………………….…...34
2.2.1. Inhibitor synthesis and enzyme kinetics ……………….…………34
2.2.2. Enzyme purification ……………………………………...……….34
- 3 -
2.2.3. Crystallization and soaking...... …...34
2.2.4. Data collection and structure determination ……………………...35
2.3 Results ……………………………………………………………………….36
2.3.1. Ceftazidime BATSI structure ………………………….…………38
2.3.2. Cefoperazone BATSI structure …………………………….…….39
2.3.3. Compound 1 and compound 2 structures …………………..…….40
2.4 Discussion ………………………………………………………….…..……41
Chapter 3 Crystal Structure of SHV-1 β-Lactamase in Complex with Penem and penam sulfone inhibitors that form cyclic inhibitory intermediates …………………….58
3.1 Introduction …………………………………………………………….……58
3.2 Materials and Methods ……………………………………………...……….60
3.2.1. Enzyme purification ………………………………………….…...60
3.2.2. Crystallization and soaking ...... …...60
3.2.3. Data collection and structure determination …………………...…60
3.3 Results and Discussion ……………………………………………..……….62
3.3.1. SHV-1: Penem 1 structure …………………………………..……62
3.3.2. SHV-1: SA1-204 structure ………………………………………..64
Chapter 4 Trans-Enamine Intermediate Formation as a β-Lactamase Inhibition Strategy Probed for Inhibitor-Resistant S130G SHV β-Lactamase...... 76 - 4 -
4.1 Introduction ...... …...76
4.2 Materials and Methods ………………………………………………………78
4.2.1. Inhibitors...... …....78
4.2.2. Mutagenesis, purification and crystallization …………………….78
4.2.3. Soaking, data collection and structure determination …………….78
4.3 Results ...... …...79
4.4 Discussion ……………………………………………………………..…….80
Chapter 5 Crystal Structure of KPC-2 β-Lactamase ………………………………..….88
5.1 Introduction …………………………………………………………….……88
5.2 Materials and Methods ………………………………………………………90
5.2.1. Enzyme expression and purification ……………………………...90
5.2.2. Crystallization ………….………………………………………....91
5.2.3. Data collection and structure determination …………………..….91
5.3 Results …………………………………………………………………….....93
5.4 Discussion ……………………………………………………………...……95
Chapter 6 Crystal Structure of KPC-2 β-Lactamase in Complex with 3-NPBA and PSR3-226 ………………………………………………………………...……….114
6.1 Introduction ………………………………………………………………...114
- 5 -
6.2 Materials and Methods ……………………………………………….…….115
6.2.1 Subcloning ………………………………………………….…....115
6.2.2 Expression and purification ……………………………………...116
6.2.3 Crystallization and soaking ………………………………………117
6.2.4 Data collection and structure determination ……………………..118
6.3 Results and Discussion……………………………………………………..118
6.3.1 KPC-2: 3-NPBA structure …………………………….…………118
6.3.2 KPC-2: PSR3-226 structure ……………………………….……..120
6.4 Conclusion...... …...122
Chapter 7 Heterotropic Allosteric Regulation Mechanism of an Engineered β- Lactamase-MBP Fusion Protein………………………………………………………..131
7.1 Introduction ……………………………………………………..…………131
7.2 Materials and Methods …………………………………………………….133
7.2.1 Expression and purification ………………..…………………….133
7.2.2 Crystallization and soaking ……………………………….……...134
7.2.3 Data collection and structure determination ……………..………134
7.2.4 Molecular Dynamics Simulation ………………………….……..136
7.3 Results ………………………………………………………………….…..137
- 6 -
7.3.1 Overall structure ………………………………………………….137
7.3.2 Zinc binding ………………………………………………….…..139
7.3.3 MBP domain and TEM-1 domain ………………………….…….140
7.3.4 Mutagenesis………………………………………………….…...142
7.3.5 Molecular dynamics simulations of RG13……………………….143
7.4 Discussion…………………………………………………………….……144
7.4.1 Insights into maltose activation of RG13…………………..….145
7.4.2 General applicability of MBP as a sensory allosteric domain in fusion constructs……………………………………………………..149
7.4.3 Serendipitous zinc binding and regulation in RG13………..….151
Chapter 8 Summary and Future Directioins …………………………………………..174
8.1 Summary ………………………………………………………...…………174
8.2 Future directions …………………………………………………..……….180
Bibliography …………………………………………………………………………185
- 7 -
LIST OF TABLES
Table 2.1 Inhibition data for BATSI compounds…………………………………..56
Table 2.2 Data collection and refinement statistics…………………...……………57
Table 3.1 Data collection and refinement statistics………………………...………75
Table 4.1 Data collection and refinement statistics………………………….……..87
Table 5.1 Data collection and refinement statistics for KPC-2 structure………….111
Table 5.2 Active site distance changes of carbapenemases and non-carbapenemases...... …...112
Table 5.3 Relative shifts in active site residues of carbapenemases compared to non-carbapenemases……………………………………………………………………113
Table 6.1 Inhibition and kinetics data………………..……………………………129
Table 6.2 Data collection and refinement statistics ……………………………....130
Table 7.1 Data collection and refinement statistics...... …...172
Table 7.2 Kinetics data from the literature……………………………..…………173
- 8 -
LIST OF FIGURES
Figure 1.1 Defense mechanisms of gram-negative bacteria against β-lactam antibiotics and β-lactamase inhibitors……………………………………...…..28
Figure 1.2 Typical structures in the β-lactam family are listed including the representative penicillin, cephalosporin, carbapenems and β-lactamase inhibitors ………………………………………………………………………….……..29
Figure 1.3 Crystal structure of SHV-1 β-lactamase (PDB ID: 1SHV)……… …..…30
Figure 2.1 Chemical structures of compounds and the synthesis scheme ...... …...49
Figure 2.2 Unbiased omit Fo-Fc maps……………………………………...……….50
Figure 2.3 Stereo view of interactions of the bound ligands within SHV-1 β-lactamase active site………………………………………………………………….. 51
Figure 2.4 Comparisons of β-lactamases and their bound BATSIs… …………...…53
Figure 3.1 Chemical structures and reaction schemes ……………………………...68
Figure 3.2 Electron density maps are depicted ……………………………………..70
Figure 3.3 Stereo view of interactions of the bound ligands within SHV-1 β-lactamase active site…………………………………………………………………...72
Figure 3.4 Structural superpositions ………………………...………………………73
Figure 4.1 Chemical structures of Clavulanic acid, tazobactam and SA2-13 and proposed reaction scheme of a generalized inhibitor with Class A β-lactamases based on previous work …………………………………………………………………….…..82
Figure 4.2 Electron density of the SA2-13 compound in the active site of S130G SHV-1 β-lactamase …………………………………………….………………………..83
- 9 -
Figure 4.3 Stereo view of interactions of SA2-13 within S130G SHV β-lactamase active site……………………………………………………………………...…………84
Figure 4.4 All Cα superposition of SA2-13-S130G SHV with SA2-13-SHV-1……85
Figure 5.1 Schematic diagram of penicillin G, a carbapenem (imipenem), cephamycin (cefoxitin), a cephalosporin (cephalothin), and bicine………………..…..104
Figure 5.2 Electron density map………………………………..…………………..105
Figure 5.3 Structure of KPC-2 β-lactamase…………………………...………...…106
Figure 5.4 Stereo figure depicting the active site of KPC-2 with bicine and a superpositioned cephalosporin and schematic diagram of interactions of bicine in the active site of KPC-2………………………………………………………………….…107
Figure 5.5 Superposition of class A carbapenemases and the active sites of the carbapenemases and non-carbapenemase β-lactamases …………….…………………108
Figure 5.6 Active site adjustments of KPC-2 and their postulated role in accommodating cefoxitin……………………………………………………………….109
Figure 6.1 Chemical structures of the inhibitors and the proposed reaction scheme ……………………………………………………………………………..…. 123
Figure 6.2 Unbiased omit Fo-Fc maps contoured at 2.5σ are depicted ………………………………………………………….……………………..………. 124
Figure 6.3 Stereo view of interactions of the bound inhibitors within KPC-2 β- lactamase active site…………………………………………………………………….126
Figure 6.4 Active site superposition of KPC2:3-NPBA structure with SHV- 1:Cefoperazone BATSI structure (PDB ID: 3MKF) and active site superposition of KPC2:PSR3-226 structure with SHV-1:SA2-13 structure (PDB ID: 2H5S)……….….127
Figure 7.1 Overal structure of RG13….……………………………………….…. 154
- 10 -
Figure 7.2 Active site and linker regions of RG13 ………………………………...155
Figure 7.3 Zinc binding sites in RG13……………………………………………..156
Figure 7.4 Structural analysis of the MBP subdomain angle variations…………...157
Figure 7.5 Electron density map showing active site β3 strand displacement in TEM-1 domain of RG13……………………………………..…………………………158
Figure 7.6 Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure …………….…………………..……………………………………. 159
Figure 7.7 R.m.s.d. of backbone atoms calculated relative to the starting structure for RG13 and TEM-1 molecular dynamics simulations …………….……………...….161
Figure 7.8 Molecular dynamics simulations reveals zinc dependent changes in relative orientation of MBP and TEM-1 domains ……………………………………..163
Figure 7.9 Modeling of RG13 fusion protein with a catalytically competent TEM-1 domain near MBP fusion site to relay maltose-induced conformational changes …………………………………………...………………………………….....164
Figure 7.10 The Cα-Cα difference plot for maltose-free and maltose-bound MBP………………………………………………………….…………………………166
Figure 7.11 Surface depiction of RG13 showing potential zinc liganding surface residues ……….………………………………………………………………………..167
Figure 7.12 Regulatory zinc binding sites in RG13. RG13 (yellow and magenta) is superimposed onto wt TEM-1 (in grey)………….……………………………….….168
Figure 7.13 Schematic diagram of heterotropic allosteric regulation of RG13 by maltose and zinc …………….…………………………………...…….……………170
Figure 8.1 Proposed reaction pathway of an ineffective inhibitor being hydrolyzed based on previous work and Chemical structures of three clinical used β-lactamase inhibitors and of inhibitors that have been discussed in this thesis work.……………...181
- 11 -
Figure 8.2 Elution chromatogram of KPC-2 E166A on the Superdex 200 column ………….……………………………………………………………………………….183
Figure 8.3 Clusters of RG13 in the presence of maltose ……………………….….184
- 12 -
ACKNOWLEDGEMENTS
I am deeply indebted to my mentor, Dr. Focco van den Akker, for the freedom,
encouragement, enthusiasm, inspirations and support he gave me during my whole Ph.D
studies. I learned countless lessons from him not only about science but also about life. I
was extensively trained to improve English, technical skills, critical thinking abilities,
the way to organize slides and the way to present my scientific results. I feel honored to
be his graduate students and friend as well.
I want to express my special thanks to Dr. Vivien Yee. She has been such a
thoughtful and caring people I ever met. She provided lots of scientific insights during
joined group meeting. And she also shared lots of her experiences to be a mother. I
would also like to thank all the previous and current group members in both Focco’s lab
and Vivien’s lab. I enjoyed the time of joined group meetings and joined group
activities.
I would like to thank the members of my advisory committee for their helpful discussion and advices. I would like to thank our collaborators for providing the inhibitors, kinetic data and the enzyme construct. I would like to thank beamline support in NSLS, SSRL, APS, and the in-house Rigaku system in Pharmacology department for data collection. I would like to thank Department of Biochemistry at Case Western
Reserve University for giving the great study and research environment. There are many staff members at this department and university, to whom I’d like to say “Thank you”
- 13 - too.
I would like to thank my friends Yi, Yuanyuan, Lu, and all the other friends as well.
It has been such a great time to play together, do researches together, to discuss
problems met and to share all the feelings encountered.
Finally, I would like to thank my parents, my sister, my husband and my daughter
for their tremendous help and support. I would like to say that without you I could not go
through all the way to my thesis defense.
- 14 - LIST OF ABBREVIATIONS
AmpC, AmpC cephalosporinase (a class C β-lactamase)
APS, Advanced Photon Source
BATSI, boronic acid transition state inhibitor
CTX-M, active on Cefotaxime, first isolated at Munich (a class A β-lactamase)
ESBL, extended spectrum β-lactamases
IC50, the half maximal inhibitory concentration
IPTG, isopropyl β-D-1-thiogalactopyranoside
IR, inhibitor resistant
KPC, Klebsiella pneumoniae carbapenemase
MIC, minimum inhibitory concentration
NMC-A, not metalloenzyme carbapenemase A
3-NPBA, 3-nitrophenyl boronic acid
NSLS, National Synchrotron Light Source
OMP, outer membrane protein
PDB, Protein Data Bank
PEG, polyethylene glycol r.m.s.d., root mean square deviation
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis
SHV, sulfhydryl reagent variable, describing a property of the β-lactamase
SME, isolated from Serratia marcescens S6 and designated Serratia marcescens enzyme
SSRL, Stanford Synchrotron Radiation Lightsource
- 15 - TEM, β-lactamase named after the patient Temoneira providing the first sample
WT (or wt, or wt), wild-type
- 16 - Mechanisms of β- lactamase Inhibition and Heterotropic Allosteric
Regulation of an Engineered β- lactamase-MBP Fusion Protein
Abstract
by
WEI KE
Expression of β-lactamases is the most common mechanisms of β-lactam antibiotic resistance clinically encountered. Both the numbers and types of β-lactamases are growing rapidly. Therefore, there is an urgent need to develop more potent antibiotics and β-lactamase inhibitors. My thesis work was focused on exploring the structural basis of β-lactamases overcoming new β-lactam antibiotics and inhibitors, on exploring this knowledge to develop improved inhibitors, and on exploring other ways of regulation
(molecular switch). The SHV-1 β-lactamase, KPC-2 carbapenemase and inhibitor- resistant S130G SHV will be used as the model system to characterize the active site chemistry of enzyme-inhibitor complexes. Three types of inhibitors were tested including the ones exploiting the function of R1 substituent, exploiting the function of R2 substituent, and exploiting transition state inhibitors. Also TEM-MBP fusion protein will be used to investigate the mechanisms of heterotropic allosteric regulation.
- 17 -
CHAPTER 1
Background and Significance
1.1 Antibiotic Resistance Crisis
Various antimicrobial agents were developed to treat bacterial infections by inhibiting cell wall synthesis, protein synthesis or DNA replication (Neu, 1992). Efforts have been dedicated to develop antibiotics targeting bacterial cell wall since the last century because such compounds could potentially have limited toxic side-effects as humans do not share the same proteins. These latter antibiotics targeting the penicillin binding proteins (PBP) are called β-lactam antibiotics; their characteristic feature is that they all contain a β-lactam core structure. β-lactam antibiotics mimic the natural substrate of PBP D-Ala-D-Ala peptide (Knox et al., 1996); they covalently inhibit PBP therefore blocking it from cross-linking and thereby weakening the bacterial cell wall thus leading to the cell lysis and death.
To counteract the lethal effect of β-lactam antibiotics, bacteria have developed four major resistance mechanisms: expression of β-lactamases that degrade β-lactam antibiotics; mutant PBPs that have a decreased the affinity for β-lactam antibiotics; mutant OMP (outer membrane protein) proteins or loss of OMPs that reduce uptake into the cell (Drawz, Bonomo, 2010;Davies, 1994;Thomson, Bonomo, 2005); and increased expression of efflux pumps that remove antibiotics out of the cell. Among these resistance mechanisms, expression of β-lactamases is the most widespread mechanism in gram negative organisms and is often the most efficient one at overcoming antibiotic
- 18 - pressures by β-lactam antibiotics (Yang et al., 1999;Matagne et al., 1998;Matagne et al.,
1999;Livermore, 1995;Fisher et al., 2005).
Selection pressure by antibiotics constantly challenges bacteria to evolve and resistance mechanisms usually exist before their use in clinic. Not long after the discovery of penicillin, the first penicillinase was reported. 1n 1944, benzylpenicillin was first used therapeutically and was active against 95% of S. aureus isolates while five years later this number was reduced to 50%. New types of β-lactam antibiotics were developed which included cephalosporins and carbapenems. β-lactamase inhibitors were used as well which are used in combination with β-lactam antibiotics. However, to survive, bacteria soon evolved to synthesize extended spectrum β-lactamases (ESBL) to counteract cephalosporins, carbapenemases to counteract carbapenems and inhibitor- resistant β-lactamases (IR) to counteract β-lactam antibiotic/β-lactamase inhibitor combinations (Coulthurst et al., 2005) (Figure 1.1). Early in the 1970s, only a handful β- lactamases were known; now the number exceeds 920 (personal communication Dr.
Bonomo). β-lactamases are increasing at an alarming rate and antibiotic resistance caused by β–lactamase expression has been a big clinical concern (Boucher et al.,
2009;Livermore, Woodford, 2006;Nicasio et al., 2008;Paterson, Doi, 2007;Talbot et al.,
2006). There is thus an urgent need to develop novel β-lactam antibiotics and β- lactamase inhibitors.
1.2 β-lactamase overview
- 19 - There are two major classification schemes for categorizing β–lactamases: the
Ambler and the Bush-Jacoby-Medeiros classifications. The former classifies β– lactamases based on amino acid identity or similarity (Ambler et al., 1991). Now there are four classes defined. Classes A, C and D are serine-dependent β-lactamases whereas class B enzymes are metallo-β-lactamases that require one or two Zn2+ ions for activity.
The Bush-Jacoby-Medeiros scheme classifies β-lactamases into groups 1 though 4 based on functional characteristic (substrate and inhibitor profile) (Bush et al., 1995).
Moreover, certain β-lactamases follow additional nomenclature. For example, carbapenemases refer to β–lactamases that are able to degrade carbapenems. Also inhibitor-resistant variants refer to β-lactamases that are able to degrade β –lactamase inhibitors. In this study we will focus on the Class A β-lactamases SHV-1, TEM-1, carbapenemase KPC-2, and the inhibitor resistant S130G SHV variant.
1.2.1 SHV-1 and TEM-1 β –lactamases
TEM-1 and SHV-1 share 68% amino acid sequence identity and are representative Class A serine β-lactamases commonly detected in clinical isolates of E. coli and K. pneumoniae pathogens. These are clinically important organisms responsible for not only nosocomial infections but also urinary tract infections in outpatient setting
(Roy et al., 1985;Gorbach, 1994). TEM-1 is the first plasmid-mediated β-lactamase identified and named after the patient from whom it was isolated (Datta, Kontomichalou,
1965). Chapter 7 will focus on an engineered TEM-1 fused to MBP to understand its unconventional engineered allosteric regulation mechanism. SHV was named from the term “sulfhydryl reagent variable” because early studies showed that it is responsive to
- 20 - inhibition by the sulfhydryl reagent group p-chloromercuribenzoate (Matthew et al.,
1979). Chapters 2 & 3 will use SHV-1 β-lactamase as the model to study inhibitor- enzyme complex structures to elucidate the mechanisms of inhibition.
1.2.2 Inhibitor resistant (IR) β-lactamases
IR β-lactamases are challenging the current β-lactam antibiotic/β-lactamase inhibitor combinations efficacy to treat serious urinary tract, respiratory tract and bloodstream infections that are accompanied by β-lactamase-mediated resistance mechanisms (Buynak, 2006;Drawz, Bonomo, 2010;Page, 2000;Sandanayaka, Prashad,
2002). Class A IR β –lactamases are usually TEM or SHV variants having amino acid substitutions at Ambler positions 69, 130, 234, 244, 275, or 276
(www.lahey.org/studies/webt.asp). Theses IR enzymes typically have increased Ki and
IC50 values for β-lactamase inhibitors. The mechanisms of resistance to clavulanic acid is primarily attributed to the alterations of the oxyanion hole geometry and changes in the positions of S130 or R244 (Drawz, Bonomo, 2010). In Chapter 4, we will focus on an IR phenotype S130G SHV.
1.2.3 Carbapenemases
Carbapenemases seriously threaten the current antibiotic armamentarium by their ability to hydrolyze carbapenems which are often clinically considered the “last resort” antibiotics. In addition to carbapenems, carbapenemases can also hydrolyze almost all β- lactams and as such are now recognized as the most versatile family of β-lactamases with a breadth of substrate spectrum unrivaled by other β-lactamases (Queenan, Bush, 2007).
- 21 - Carbapenemases have been identified through Classes A to D β-lactamases (Queenan,
Bush, 2007;Kim et al., 2006;Walther-Rasmussen, Hoiby, 2006;Walsh et al., 2005) and are disseminated both chromosomally and on plasmids. Acquired carbapenemases were relatively rare back in 2002 but there was already significant concern of their potential rapid dissemination (Livermore, 2002). To date, outbreaks of carbapenemases have been reported worldwide and resistance caused by carbapenemases has become unfortunately a global problem (Queenan, Bush, 2007). There is, therefore, an urgent need to understand the properties of this type of β-lactamase and its mechanism of inhibition. In this study we will focus on Class A Klebsiella pneumoniae carbapenemase (KPC).
Class A carbapenemases currently have six families: GES-, KPC-, NMC-, SME-,
BIC- and SFC-type enzymes. Among the six families, only the KPC-type and GES-type carbapenemases are widely distributed in clinical bacterial pathogens (Frase et al., 2011).
The first member of the KPC family, KPC-2, was clinically isolated in 1996 in North
Carolina (Yigit et al., 2001). To date 11 KPC variants have been described
(http://www.lahey.org/studies/other.asp#table1) with KPC-2 and KPC-3 being the dominant ones and endemic in the United States, Greece and Israel (Woodford et al.,
2011). Increasingly reports are emerging from China, South America and many countries in Europe (Woodford et al., 2011). The genes encoding the KPC family of enzymes are located on transposons and transmissible plasmids which allows for rapid dissemination of the KPC gene. Although Klebsiella pneumoniae is the predominant host species for
KPC β-lactamases, Escherichia coli, Pseudomonas spp. and Acinetobacter spp. have
- 22 - recently been reported to harbor the KPC-2 gene (Woodford et al., 2011). This represents a disturbing and worrying development in the spread of these carbapenemases.
Crystal structures of apo NMC-A, NMC-1-inhibitor complex, SME-1, apo GES-2 and GES-2-inhibitors have been reported (Frase et al., 2011;Mourey et al., 1998;Swaren et al., 1998;Sougakoff et al., 2002) and contributed significantly to our understanding of the carbapenemase at the molecular level. We reported apo KPC-2 structure in 2007 (Ke et al., 2007) (Chapter 5). Dr. Sougakoff’s lab reported the apo and a C-terminal deleted version of KPC-2 one year later (Petrella et al., 2008). In chapter 6, we will describe crystal structures of KPC-2 in complex with a boronic acid transition state inhibitor and a designed trans-enamine stabilizing inhibitor. Our work will advance our understanding of the mode of action of carbapenemases and their inhibition and will have potential implications for future structure-based drug design efforts.
1.3 β-Lactam antibiotics and β-lactamase inhibitors
Different aspects of β-lactam antibiotics and β-lactamase inhibitors are well reviewed in many publications (Buynak, 2006;Drawz, Bonomo, 2010;Lee et al.,
2003;Page, 2000;Therrien, Levesque, 2000;Coates et al., 2011;Sandanayaka, Prashad,
2002). For brevity, we summerize some basic information.
There are numerous antibiotics clinically used with over 50 compounds based upon penicillin, 70 cephalosporins, five carbapenems and others (Drawz, Bonomo, 2010).
More than 20 new classes of antibiotics were marketed since the last century. β-lactam
- 23 - antibiotics have the advantages of low cost, bacterial specificity (often little toxic side- effects), high effectiveness, and ease of delivery, which makes them extremely valuable clinical resources. Facing the challenge of the resistant β-lactamases, two major strategies could be applied which include development of analogues of the current drugs or development of novel non-β-lactams which are gaining importance (Coates et al., 2011).
In this thesis, both types of inhibitors are studied.
β-lactam antibiotics and β-lactamase inhibitors are both small molecule compounds containing the core structure of a 4-membered β-lactam ring. Typical structures in this β-lactam family are listed in Figure 1.2 including representative penicillin, cephalosporin, carbapenems and β-lactamase inhibitors. Penicillins usually are penams and have a 4, 5-fused ring with a varied R1 substitution at C6 position.
Cephalosporins usually are penems and have a 4, 6-fused ring with a varied bulky R1 substitution at C7 position. Additionally, cephalosporins have a varied R2 substitution at
C3 position. Carbapenems have specialized features in that they contain a β-lactam ring fused to an unsaturated five-membered carbon ring penem and also have a unique 6α substitution which renders them active against a wide variety of multi-drug-resistant and difficult-to-treat Gram-negative bacteria pathogens (Bradley et al., 1999;Maveyraud et al.,
1998). The current clinically used β-lactamase inhibitors are either penam (clavulanic acid) or penam sulfone (sulbactam or tazobactam) which all do not have any substituents at the C6 position. In this study, we investigated several β-lactamase inhibitors including a penam sulfone (SA2-13 analogue), C6-methylidene substituted penems, C6- methylidene substituted penam sulfone (a LN-1-255 analogue), and several non-β-lactam
- 24 - inhibitors called boronic acid transition state analogues, which will be discussed more in detail in the following chapters. Our studies and findings will hopefully contribute to future drug design efforts to develop new potent β-lactamase inhibitors.
1.4 Structures of Class A β-lactamases
Despite the diversity of the primary structures, Class A β-lactamases share a high degree of similarity in their tertiary structures: they all contain a well conserved active site with S70 as the catalytic residue (Frase et al., 2011;Herzberg, Moult, 1987;Jelsch et al., 1993;Nukaga et al., 2003b;Knox, Moews, 1991;Ke et al., 2007;Kuzin et al.,
1999;Swaren et al., 1998;Sougakoff et al., 2002). The four conserved elements making up the active site are (Figure 1.3): S70-X-X-K73 sequence (SXXK motif), S130-D131-
N132 sequence (SDN motif), K234 –T235 -G236 sequence (KTG motif), and the Ω-loop comprising residues from 164 to 179 (Matagne et al., 1998). S70 is the catalytic serine.
SDN motif is located in the all- α domain forming one side of the substrate-binding cavity. KTG motif is located on the α/β domain forming the opposite side of the substrate-binding cavity. The Ω-loop harbors the important residue E166. In addition, there are two critical water molecules in the active site. One is the catalytic or deacylation water molecule primed by S70, E166 and N170. The other water molecule usually temporally occupies the oxyanion hole positioned by the backbone amide nitrogens of residues 70 & 237. The position of this water molecule reflects position of the carbonyl of the β-lactam substrate during acylation and deacylation. The role of the highly conserved active site residues S70, K73, S130, E166, K234, oxyanion hole and Ω-loop in
Class A β-lactamase will be briefly summarized below.
- 25 -
S70 is the nucleophile that attacks the carbonyl carbon of the β-lactams and leads to the cleavage of the C-N bond. K73 makes a hydrogen bond with S70 and was suggested to be involved in the proton relay during acylation. S130 was suggested to participate in substrate recognition, facillitate β-lactam ring opening during enzyme acylation, and initiate the irreversible step of inactivation (Kuzin et al., 2001;Imtiaz et al.,
1994;Imtiaz et al., 1993;Atanasov et al., 2000;Vakulenko et al., 1998;Lamotte-Brasseur et al., 1991). E166 has an important role in acylation and in positioning the deacylation water molecule. Several residues have been proposed to act as the general base in acylation: K73, the E166/water pair, and the substrate carboxylate/S130 moieties
(Damblon et al., 1996;Diaz et al., 2003;Hermann et al., 2005;Meroueh et al.,
2005;Minasov et al., 2002;Chen et al., 2007;Strynadka et al., 1992;Golemi-Kotra et al.,
2004). K234 was also suggested to be an important active-site residue involved in both ground-state and transition-state binding (Lenfant et al., 1991;Ellerby et al., 1990). The oxyanion hole is a very important functional presence as it helps form the transitional species at the early stage of the acylation reaction (Curley, Pratt, 2000). The Ω-loop
(residues 164-179) is a very important structural component containing the critical residue E166. Proper positioning of this loop ensures the presence of the catalytic E166 in the active site. A recent study shows that increased flexibility of the Ω-loop could aid in explaining certain ESBL phenotypes (Sampson et al., 2011).
Although class A β-lactamases have similar tertiary structures, they display different substrate profiles and resistance characteristics with a single amino acid
- 26 - substitution (for example ESBLs or inhibitor-resistant β-lactamases) or substantially diverged primary sequence (for example carbapenemases). In this study, we will focus on the parent SHV-1, inhibitor-resistant S130G SHV and carbapenemase KPC-2, with the aim of trying to further elucidate insights into mechanisms of resistance that could be used for the development of β-lactamase inhibitors.
- 27 -
Figure 1.1. Defense mechanisms of gram-negative bacteria against β-lactam antibiotics and β-lactamase inhibitors.
- 28 -
Figure 1.2. Typical structures in the β-lactam family are listed including the representative penicillin, cephalosporin, carbapenems and β-lactamase inhibitors.
- 29 - A
B
- 30 - Figure 1.3. Crystal structure of SHV-1 β-lactamase (PDB ID: 1SHV). (A) Cartoon representation of the overall structure; (B) Active site of SHV-1 β-lactamase. Conserved
SXXK motif is colored red; conserved SDN loop is colored yellow, β3 strand including the conserved KTG motif is colored orange; and the Ω-loop is colored in blue. W1 is the deacylation water and W2 is the oxyanion water molecule and colored in magenta.
Oxyanion hole is box-highlighted.
- 31 - CHAPTER 2
Crystal Structures of SHV-1 β-Lactamase in Complex with
Boronic Acid Transition State Inhibitors
(Ke et al., 2011)
2.1 Introduction
Production of β-lactamases (E.C.3.5.2.6) is one of the major mechanisms by which bacteria develop resistance to β-lactam antibiotics. In nature, four classes of β- lactamase enzymes exist (classes A-D). Class A, C and D are serine-based β-lactamases, while class B uses a metal ion (Zn2+) to hydrolyze the lactam bond. TEM-1 and SHV-1 β- lactamases are among the most commonly observed Class A β-lactamases found in
Escherichia coli and Klebsiella pneumoniae. As such, this family of β-lactamases presents a significant clinical threat (Bush, 2002;Paterson, Bonomo, 2005).
To counteract β-lactamases, mechanism-based inhibitors were developed to be administered in concert with β-lactam antibiotics (Drawz, Bonomo, 2010). Presently, there are three commercially available β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam, Figure 2.1), which are effective primarily against class A β-lactamases.
Unfortunately, bacteria possessing β-lactamase enzymes have adapted under continued drug pressure and some variants of class A are now “inhibitor resistant” (Drawz,
Bonomo, 2010). Moreover, class C and D enzymes are poorly inhibited by any of the three commercial inhibitors. Therefore, there is an urgent need to develop novel β-lactam antibiotics and new types of β-lactamase inhibitors to counteract the current crisis in antimicrobial resistance (Drawz, Bonomo, 2010).
- 32 - Based upon properties distinctive to the boron atom, boronic acid transition state inhibitors (BATSIs) were developed as serine β-lactamase inhibitors (Crompton et al.,
1988;Kiener, Waley, 1978). By being competitive and reversible inhibitors, boronates also offer an opportunity to study the reaction coordinate (mechanism) of a β-lactam
(Thomson et al., 2007b) interacting with a β-lactamase.
In the development of these active site probes, one approach uses the R1 side chains of potent β-lactam antibiotics and combines them with a boronic acid moiety with the goal to mimic the transition state and create a high affinity, reversible inhibitor that cannot be inactivated by β-lactamases, due to not bearing the hydrolyzable β-lactam ring
(Strynadka et al., 1996). In this context, the efficacy and mechanism of inhibition of
BATSIs has been explored against a variety of clinically important β-lactamases. Most of the studies have been focused on TEM-1 and AmpC β-lactamases with a few studies exploring the inhibition of CTX-M family as well (Powers et al., 2001;Minasov et al.,
2002;Chen et al., 2005). Although a number of functional studies have been performed on the mechanism of inhibition of BATSIs against inhibitor resistant and extended- spectrum SHV β-lactamases (Drawz et al., 2009;Thomson et al., 2006;Thomson et al.,
2007b), structural analyses have not yet fully complemented these previous investigations which is the primary focus of this study.
We present kinetic and crystallographic data describing different BATSIs inhibiting SHV-1 β-lactamases. These BATSIs include a ceftazidime-BATSI and a cefoperazone-BATSI each containing the R1 side chain of their respective β-lactam
- 33 - antibiotic. In addition, we have synthesized and characterized structure-based derivatives of the latter BATSI. These new BATSI:SHV-1 structures and BATSI derivatives expand our understanding of the mechanism of inhibition against a clinically important family of
β-lactamases and yield gains and insights for future β-lactamase inhibitor design.
2.2 Materials and Methods
2.2.1 Inhibitor synthesis and enzyme kinetics.
The BATSIs were synthesized by Dr. Prati’s lab. The enzyme kinetics were provided by Dr. Bonomo’s lab (Ke et al., 2011).
2.2.2 Enzyme purification
SHV-1 β-lactamase was expressed and purified as previously described (Padayatti et al., 2006). Briefly, E. coli DH10B cells harboring the blaSHV-1gene of SHV-1 β- lactamase were grown overnight in lysogeny broth containing 20µg/mL chloramphenicol.
Cells were pelleted and lysed by stringent periplasmic fractionation. The soluble fraction, after preparative isoelectric focusing and gel filtration using Superdex75 column, yielded homogeneous SHV-1 protein as demonstrated by SDS-PAGE. SHV-1 β-lactamase in
2mM HEPES buffer pH 7.0 was concentrated to 5mg/mL using the Bradford assay.
2.2.3 Crystallization and soaking
Crystals were grown at 20°C using vapor diffusion sitting drop technique (Kuzin et al., 1999). A 5µL drop was prepared using 2.0µL protein solution, 0.5µl 5.6mM
Cymal-6 (Hampton Research), and 2.5µL reservoir solution (20-30% PEG6000 in
- 34 - 100mM HEPES pH7.0) and equilibrated over a 1ml reservoir solution. Crystals of SHV-1 grew in 2-3 days. SHV-1 β-lactamase crystals were soaked with mother liquor containing
10% (v/v) saturated ceftazidime-BATSI for 1 hour to obtain the ceftazidime-BATSI bound structure (This soaking was done by Jared and included for integrity purpose). To obtain the cefoperazone-BATSI bound structure, crystals were soaked in mother liquor containing 50mM cefoperazone-BATSI overnight. The same is done as cefoperazone-
BATSI for the cefoperazone-BATSI analogues except that the soaking concentration of compound 1 is 20mM and compound 2 is 25mM. The soaked crystals were cryo- protected with 20-25% 2-methyl-2,4-pentanediol (MPD) in mother liquor containing the corresponding inhibitor and flash frozen in liquid nitrogen prior to data collection.
2.2.4 Data collection and structure determination
X-ray diffraction data for the SHV-1: ceftazidime-BATSI complex was collected at the Advanced Photon Source 19-ID (This data was collected by Jared and included for integrity purpose). Data for the cefoperazone-BATSI complex was collected at the
National Synchrotron Light Source X-29. Data for the compound 1 complex and compound 2 complex were collected at the Stanford Synchrotron Radiation Lightsource
(SSRL) beam line 7-1. All of the data were processed using HKL2000 (Otwinowski,
Minor, 1997). Structures were determined using the isomorphous crystal structure of wt
SHV-1 β-lactamase complexed with tazobactam (PDB 1VM1) (Kuzin et al., 1999)
(tazobactam and waters were removed before refinement). Refinement was carried out using REFMAC (Murshudov et al., 1997) and model building was done using COOT
(Emsley, Cowtan, 2004). After initial refinement, strong density in the active site was
- 35 - observed for each of the respective BATSIs. The PRODRG2 server (Schuttelkopf, van
Aalten, 2004b) was used to obtain the parameter and topology files for the four BATSIs.
In the SHV-1/ceftazidime-BATSI complex structure, additional electron density was present in between two neighboring crystallographically related protein molecules representing an intact ceftazidime-BATSI molecule esterified with MPD. MPD was present in trace amounts in the pinacol used as a protecting group for the synthesis of ceftazidime-BATSI and ultimately leading to the formation of this six-membered cyclic ester (a slowly-hydrolysable boron ring structure that was detected by 1H-NMR in less than 5% in ceftazidime-BATSI). Crystallographic refinement was monitored using the program DDQ (van den Akker, Hol, 1999a) and the final model quality was assessed using PROCHECK (Laskowski et al., 2001). Data collection and refinement statistics are shown in Table 2.2.
Coordinates and structure factors for the SHV-1 complexes with ceftazidime-, cefoperazone-BATSI, compound 1, and compound 2 have been deposited with the PDB
(PDBid: 3MKE. 3MKF, 3MXR, 3MXS, respectively).
2.3 Results
Our kinetic studies on SHV-1 showed a large difference between the Ki values of ceftazidime-BATSI compared to those previously reported for other β-lactamases (Table
2.1). With respect to ceftazidime-BATSI, the lowest Ki was observed against AmpC (20 nM) and CTX-M-9 (15 nM) whereas the Ki against SHV-1 was about 100 fold higher
(Ki= 2.2µM). We interpret these differences to mean that despite significant protein sequence homologies among the β-lactamases and possible differences in the evaluation
- 36 - of kinetic constants , the detailed interactions of the active sites of these enzymes are likely significantly different and that improvement of these BATSIs are needed to attempt to arrive at BATSIs with inhibitory potency against a broader array of β-lactamases.
The crystal structures of BATSI ceftazidime-, cefoperazone-BATSI, compound 1 and compound 2 in complex with Class A SHV-1 β-lactamase were determined to 1.75Å,
1.33Å, 1.30Å, and 1.24 Å resolution, respectively (Table 2.2). The initial unbiased omit
|Fo|-|Fc| map of both structures revealed a covalently attached ligand to the Oγ atom of
Ser70 of SHV-1 β-lactamase with a single conformer (Figure 2.2A, 2.2B, 2.2D, 2.2E and
2.3). Including the respective BATSIs in refinement reduced the R/Rfree considerably indicating the correct placement of the inhibitor in the active sites for each of the data sets. In addition, the initial unbiased omit |Fo|-|Fc| map of the SHV-1: ceftazidime-
BATSI complex structure revealed a ceftazidime-BATSI analogue situated between symmetry related molecules (Figure 2.2C). This non-covalently bound ceftazidime-
BATSI analogue was subsequently included in refinement resulting in a further decrease in the R/Rfree.
The ceftazidime-BATSI: SHV-1, cefoperazone-BATSI, compound 1 and compound 2: SHV-1 structures are very similar to the wt structure (PDB ID: 1SHV) with r.m.s.d. 0.593, 0.585, 0.396 and 0.370 respectively, of all enzyme Cα atoms superposition and r.m.s.d. 0.306, 0.380Å, 0.400 and 0.381 respectively, of the active site superposition
(the 11 active site residues used in superposition were Ambler positions 69, 70, 73, 105,
130, 132, 166, 170, 234, 236 and 237). This suggests that the inhibitors do not alter the
- 37 - structure of SHV-1 in a global manner and also the active site main chain residues do not have significant changes.
2.3.1 Ceftazidime BATSI structure
In the ceftazidime-BATSI-SHV-1 β-lactamase structure (Figure 2.3A), a tetrahedral geometry is observed around the boron atom as expected for a transition state analog inhibitor. The O1 atom of ceftazidime-BATSI occupies the oxy-anion hole as it is hydrogen-bonded to the backbone nitrogen atoms of S70 and A237; the backbone oxygen atom of A237 is also interacting with the O1 atom. The O2 atom of ceftazidime-BATSI is observed interacting with the sidechains of E166 and N170 and this oxygen is thus residing in the space usually occupied by the deacylation water molecule present in Class
A β-lactamases. These observations indicate that ceftazidime-BATSI is in a deacylation transition state conformation (as defined in(Ness et al., 2000)) when bound to SHV-1 β- lactamase. The amide moiety of ceftazidime-BATSI is interacting with SHV-1 via the interaction between O6 atom of ceftazidime-BATSI and the side chain nitrogen atom of
N132. The carboxylate of the dimethyl carboxyl moiety of ceftazidime-BATSI resides in the carboxylate binding cavity conserved for the C4 carboxylate of the cephalosporin
(defined by S130, K234, T235 and R244 (Padayatti et al., 2006) of Class A β-lactamases) forming a salt bridge with R244 and hydrogen bonds with residues T235, S130 and
K234. In addition, ceftazidime-BATSI also makes a ring stacking interaction with Y105 via ceftazidime-BATSI’s thiazole ring. This allows the dimethyl carboxyl linker moiety to fold back into the active site and reside in the carboxyl binding pocket as mentioned
- 38 - above. Finally, ceftazidime-BATSI forms an intra-molecular hydrogen bond of the amine nitrogen N4 with atom O15.
Comparing the active sites of the ceftazidime-BATSI complex structure with that of apo SHV-1, the following protein atoms have shifted relative to the center of the active site (Figure 2.4A): in the SHV-1 structure, residue E166 moved in towards ceftazidime-
BATSI (0.6Å) and N170 moved away from it (0.3 Å) so that both are now within strong hydrogen-bonding distance of O2 of ceftazidime-BATSI. Moreover, S130 moved in 0.4
Å and T235 moved in 0.5 Å to stabilize the carboxylate tail of ceftazidime-BATSI. We also observed that Y105 moved inwards 0.5 Å and shifted laterally slightly to allow stacking with the thiazole ring of ceftazidime-BATSI. Residue A237 moved inwards 0.3
Å to stabilize O1 atom of ceftazidime-BATSI. Shifts less than 0.2Å are not discussed since they are close to the coordinate error.
2.3.2 Cefoperazone BATSI structure
Cefoperazone-BATSI also adopts a deacylation transition state conformation in
SHV-1 β-lactamase as it adopts the same tetrahedral geometry around the boron atom with the positions and interactions of the O1 and O2 atoms similar to those of ceftazidime-BATSI (Figure 2.3B). The amide moiety of cefoperazone-BATSI is observed interacting with the main chain carbonyl oxygen of A237 and the side chain nitrogen atom of N132. The cefoperazone R1 side chain is partially pointing toward the bulk solvent yet the phenol moiety of cefoperazone-BATSI still makes favorable end-on stacking interactions with Y105 whereas the O20 and N23 atoms of cefoperazone-BATSI
- 39 - hydrogen bond to the side chain hydroxyl group of T167. Finally, there is a water mediated interaction between O22 of cefoperazone-BATSI and the hydroxyl group of
Y105.
Comparing the active sites of the cefoperazone-BATSI complex structure with that of apo SHV-1, we see the following protein atoms moving upon binding cefoperazone-BATSI (Figure 2.4A). Like with the ceftazidime-BATSI complex, residue
E166 is shifted inwards 0.6Å towards cefoperazone-BATSI so that it is now in strong hydrogen-bond distance with O2 atom of cefoperazone-BATSI. Cα of A237 moved in
0.3Å such that carbonyl oxygen of A237 is now in hydrogen-bond distance with the N4-H of cefoperazone-BATSI whereas Y105 flipped 115º to make end-on stacking with the piperazine ring of cefoperazone-BATSI. Finally, residues S130 moved in 0.5 Å, and
T235 moved in 0.5 Å although they provide no direct interactions with cefoperazone-
BATSI.
2.3.3 Compound 1 and compound 2 structures
Compound 1 and compound 2 are analogs of the cefoperazone-BATSI and were designed to both maintain the favourable interactions (i.e. with the piperazine-containing ring) yet lessen the impact that the parent BATSI has on the Y105 conformation which is in the swung-out conformation in the starting structure of cefoperazone-BATSI. An additional design consideration was to increase the length of the phenol moiety such that future modifications of this aromatic ring can include a carboxyl moiety that can reach the conserved carboxyl binding pocket in β-lactamases. Our structure-based design
- 40 - yielded compounds 1 and 2 which have two and one additional methyl groups, respectively, in the phenol-linker with the aim to make the phenol moiety more flexible such that the end-on stacking interactions with the swung-out Y105 conformation would be disfavored. The structures of BATSIs compounds 1 and 2 reveal that both compounds adopt a deacylation transition state conformation in SHV-1 β-lactamase similar to the parent BATSI. The major portion of the designed BATSI adopts the same conformation as the parent cefoperazone-BATSI. There are some minor differences in the position of the piperazine-containing moiety although this moiety is largely in the same location
(Figure 2.4G). However, the phenol moiety of compound 1 and compound 2 are less ordered and in different positions and not end-stacked with Y105. The conformation of
Y105 has been carefully examined and has been refined as two conformations with one position similarly taken by the wt SHV-1 (0.4 occupancy) and the other position similarly taken by cefoperazone BATSI complexed SHV-1 (0.6 occupancy; Figure 2.4G). Both
Y105 and the phenol moiety of the two compounds display flexibility as shown in the omit |Fo|-|Fc| (Figure 2.4D and 2.4E). Both compounds have an increased affinity compared to the parents BATSI with compound 2 having a ~ 2 fold improvement of the
Ki value (Table 2.1).
2.4 Discussion
The four BATSI SHV-1 complex structures offer key insights into inhibition of the SHV-1 β-lactamase. We compared the ceftazidime-BATSI: SHV-1 structure with previously determined structures of ceftazidime-BATSI in complex with TEM-1, AmpC, and CTX-M (Figure 2.4B). The active site superposition reveals that ceftazidime-BATSI
- 41 - adopts a similar conformation in the active site of TEM-1, AmpC and CTX-M β- lactamase but is in a different conformation when bound in the active site of SHV-1 β- lactamase. These striking differences in BATSI orientation are especially surprising since
SHV-1, CTX-M, and TEM-1 are class A enzymes and share significant sequence identity and have very similar active sites (superpositioning of 11 active site residues of the ceftazidime-BATSI-SHV-1 structure with those in the ceftazidime-BATSI: TEM-1 structure results in an r.m.s.d. for Cα atoms of 0.299Å , Figure 2.4B). Since the
Escherichia coli AmpC is a Class C β-lactamase, where more substantial differences could be expected due to the lower sequence identity and larger active site differences, we will compare our SHV-1 complex to the more similar Class A TEM-1 and CTX-M β- lactamases. This analysis points to five major differences.
Firstly, in the complexed TEM-1 and CTX-M-9 structures, ceftazidime-BATSI behaves as an acylation transition state inhibitor with one of the boronic acid oxygen atoms in the oxy-anion hole and the other taking the extrapolated position of the lactam nitrogen in the anticipated Michaelis-Menten complex. In contrast, the ceftazidime-
BATSI:SHV-1 complex reveals that the ceftazidime BATSI is bound in a deacylation transition state conformation with one of the boronic acid oxygens occupying the deacylation water pocket (Figure 2.4B, 2.4C, and 2.4D). Secondly, while in the complexed TEM-1 and CTX-M structures, the torsion angle of atom O6-C5-C7-N14 is clinal while in the SHV-1 structure this angle is periplanar. Thirdly, in the complexed
TEM-1 and CTX-M structures, ceftazidime-BATSI’s amide nitrogen makes a weak hydrogen bond with the main chain carbonyl oxygen of A237, while in the SHV-1
- 42 - complex the 4.4 Å distance between these two atoms is too great to facilitate a hydrogen bond. Fourthly, the thiazole ring of ceftazidime-BATSI in TEM-1 and CTX-M is oriented towards and interacts with the side chain of E/D240 via its amino group. In the SHV-1 structure, ceftazidime-BATSI’s thiazole ring adopts a different orientation from that seen in the TEM-1 and CTX-M structures and this moiety is mainly stabilized by the ring stacking interaction with Y105. Finally, in complex with TEM-1 and CTX-M, ceftazidime-BATSI’s carboxylate tail points towards the bulk solvent and is mainly stabilized by water-mediated hydrogen bonds to the other parts of the inhibitor molecule
(Figure 2.4). In contrast, in the SHV-1 complex structure, ceftazidime-BATSI’s carboxylate tail folds back into the active site by positioning itself in the β-lactam carboxylate binding pocket (comprised of R234, T235, S130, and K244) in close proximity to the boron head group. Taken together, these differences indicate that ceftazidime-BATSI adopts very different orientations in the enzyme active site in the two structures. These differences of ceftazidime-BATSI conformation are accompanied by several subtle active site differences between the TEM-1/CTX-M-9 ceftazidime-BATSI complexes and the SHV-1:ceftazidime-BATSI complex structures. These active site differences between these class A β-lactamases include: (i) a movement of E166 (Cα shift of 0.7Å) towards ceftazidime-BATSI to make a hydrogen bond with one boronic acid hydroxyl group of ceftazidime-BATSI; and (ii) a movement of A237 (Cα shift of 0.5Å) such that it can no longer form a hydrogen bond with N4 of ceftazidime-BATSI (Figure
2.4E). In the SHV-1 structure, N170 moves away from ceftazidime-BATSI to make space for the second boronic acid hydroxyl group of ceftazidime-BATSI and enable a strong hydrogen bonding interaction (2.6Å).
- 43 - In the complexes with TEM-1 and CTX-M, the second boronic acid hydroxyl group of ceftazidime-BATSI takes the position close to the hydroxyl group of S130 and is stabilized by two water molecules. The latter position of this second hydroxyl group of ceftazidime-BATSI is likely one of the steric reasons why the carboxylate moiety of ceftazidime-BATSI, in the TEM-1/CTX-M structures, cannot occupy the carboxylate binding pocket the way ceftazidime-BATSI does in the SHV-1 structure.
Interestingly, ceftazidime-BATSI adopts two different orientations in the seemingly very similar TEM-1 and SHV-1 β-lactamases. Considering the entropic relationships evident here, it would seem energetically favorable for ceftazidime-BATSI to occupy the deacylation water pocket as it would release an ordered water molecule thereby increasing entropy upon ceftazidime-BATSI binding (Ness et al., 2000).
Nevertheless, the Ki values (Table 2.1) suggest that SHV-1 is an "outlier" in that it is not as readily inhibited by BATSIs compared to TEM-1 and AmpC. Although ceftazidime-
BATSI is a broad spectrum inhibitor of both Class A and Class C β-lactamases, it is about 10-fold more potent against TEM-1 and ADC and about 100 fold more potent against CTX-M-9 and AmpC β-lactamases than against SHV-1 (Table 2.1). A similar trend is also present for cefoperazone-BATSI as its inhibition of SHV-1 is also less potent compared to ADC (Table 2.1). Note that, although ceftazidime-BATSI has an unreactive 6-membered boron-ring analog of ceftazidime-BATSI present in solution as evidenced by the electron density (Figure 2.2B), this species exists only in trace amounts
(less than 5%) and so it should have little effect on the Ki value and not affect the true ceftazidime-BATSI binding in the active site. A possible reason for SHV-1 to be less
- 44 - inhibited by BATSIs could be that both ceftazidime-BATSI and cefoperazone-BATSI resulted in the deacylation transition state conformation in SHV-1 compared to the other class A β-lactamases which have BATSI’s in the acylation transition state (Figure 2.4F).
Note that adopting this deacylation transition state conformation does not necessarily indicate weaker inhibition as the chiral penicillin BATSI analogues, among the most potent BATSI inhibitors, are also observed to be in the deacylation transition state conformation (Figure 2.4F) (Ness et al., 2000;Strynadka et al., 1996). Furthermore, the potent chiral cephalothin-BATSI is observed to be in the deacylation transition state conformation (Chen et al., 2005)(Figure 2.4F).
Why does ceftazidime-BATSI adopt a deacylation transition state conformation?
In our crystal structure we observe that the carbonyl oxygen of A237 in the complex form is positioned deeper into the active site in SHV-1 compared to TEM-1 and CTX-M-9 complexes. In fact, this is also true when the apo structures of SHV-1 and TEM-1 are superimposed which indicates the retracted position of the A237 carbonyl in SHV-1 is not induced by binding of the ligand. This carbonyl oxygen is in close proximity (< 3Å) to the boronic acid oxygen atom that occupies the oxy-anion hole. Therefore, the A237 carbonyl oxygen, depending on its position, could direct the way in which the BATSIs are attacked by the S70 residue or influence the repositioning of the boron oxygens, via rotation around the boron–serine bond, after the BATSI is covalently attached to S70.
Alternatively, the different conformations of the BATSIs could also be due to the much higher ionic strength and pH that was needed to obtain the TEM-1/CTX-M-9 structures
(Minasov et al., 2002;Chen et al., 2005;Caselli et al., 2001) .
- 45 - The SHV-1: cefoperazone-BATSI structure reveals a deacylation transition state conformation of the boronic acid oxygens similar to that observed in the SHV-1: ceftazidime-BATSI structure (Figure 2.4F). The amide group of cefoperazone-BATSI interacts with both N132 and the carbonyl atom of A237 in the same fashion observed in some of the TEM-1 and CTX-M-9 BATSI complexes. An interesting feature of the cefoperazone-BATSI complex is that residue Y105 of SHV-1 rotates out such that the phenol moiety of cefoperazone-BATSI can provide an end-on interaction with Y105.
Such end-on stacking between two aromatic groups was found to be energetically favorable (Burley, Petsko, 1985). This suggests that the conformation of Y105 is flexible to a certain degree which can be taken into account in further BATSI design (Bethel et al., 2006). In addition to the orientation of Y105, the positions of the boronic acid hydroxyls are another parameter that can potentially be altered by changing the substituents of the BATSI compounds and optimized in the design of future, more potent
BATSIs.
To optimize the cefoperazone-BATSI for improved inhibitory potency against
SHV-1 and other β-lactamases, we modified the BATSI such that the favorable active site interactions with the piperazine-containing moiety were left intact yet the BATSI’s phenol linker length was increased. The reasoning for the latter modification was two- fold: to weaken the phenol moiety’s end-on stacking interaction with the Y105 in the displaced conformation and, secondly, the increased linker length of the phenol moiety could aid future iterations of this BATSI such that a carboxyl containing moiety can be added to this aromatic ring that might reach the conserved carboxyl binding pocket.
- 46 - Utilization of this β-lactamase carboxyl binding pocket by BATSI has led to some of the most potent BATSIs (Figure 2.4F) (Ness et al., 2000;Strynadka et al., 1996). Our design goal was in part successful for several reasons. First, both new BATSIs compounds 1 and
2 held the Y105 side chain less rigidly in place in the outward conformation as two weaker occupied Y105 positions were observed (Figure 2.2D and 2.2E). Secondly, both designed BATSI have an improved inhibitory potency likely a result of the release of this strained interaction that caused a partial distortion of the active site via Y105’s repositioning in the cefoperazone-BATSI complex. Third, for future BATSI design, molecular modeling using the compound 1 BATSI structure indicates that the extended and more flexible phenol moiety can be rotated towards the active site such that an additional carboxyl moiety added to the aromatic ring could reach the conserved carboxyl pocket. This and other modifications will be targeted in our future studies.
In summary, we have determined the crystal structures of two BATSI compounds and two derivatives bound to SHV-1. Our results suggest that the conformations of the
R1 side chains as observed in our BATSI structures may reveal other novel interactions compared to the R1 group of the β-lactam it mimics. For example, here we see that the carboxyl binding pocket used for the C3 carboxyl group is occupied by the R1 carboxyl moiety of ceftazidime-BATSI. This suggests that there could be differences between the
Ki of the BATSIs and the Km of the corresponding substrates, something that has been previously noted (Caselli et al., 2001). The importance of these differences remains to be explored.
- 47 - Our structures also point to interesting differences between the BATSI conformations when bound to SHV-1 and those observed with identical and similar
BATSIs bound to related β-lactamases. As drug-resistant enzymes continue to evolve, the number of parameters involved in the design of novel inhibitors will continue to grow.
We have characterized several different specific interactions between ceftazidime- and cefoperazone-BATSIs and SHV-1 that can be used as additional pieces of scaffolding in the framework for the further development and optimization of new BATSI compounds targeting β-lactamases(Drawz, Bonomo, 2010), and hopefully lead to more potent and increasingly effective broad spectrum β-lactamase inhibitors.
- 48 -
Figure 2.1. Chemical structures of compounds and the synthesis scheme. A) Chemical structures of the three clinical inhibitors tazobactam, clavulanic acid and sulbactam; chemical structures of ceftazidime and cefoperazone as well as their respective BATSIs bearing their R1 side chain: ceftazidime-BATSI and cefoperazone-BATSI; chemical structures of compound 1 and compound 2 which are the derivatives of cefoperazone-
BATSI. B) Synthesis scheme for compounds 1 and 2.
- 49 -
Figure 2.2. Unbiased omit Fo-Fc maps are depicted (A) Electron density contoured at
2.5σ of ceftazidime-BATSI in the active site of SHV-1 β-lactamase; (B) Electron density contoured at 2.5σ of cefoperazone-BATSI in the active site of SHV-1 β-lactamase; (C)
Electron density contoured at 2.5σ of the second unreacted ceftazidime-BATSI away from the active site of SHV-1 β-lactamase; (D) Electron density contoured at 2σ of Y105 residue and compound 1 in the active site of SHV-1 β-lactamase ; (E) Electron density contoured at 2σ Y105 residue and compound 2 in the active site of SHV-1 β-lactamase.
Ligand carbon atoms are colored black. Nitrogen atoms are colored gray20, oxygen atoms gray40, sulfur atom gray60 and boron atoms gray70. SHV-1 β-lactamase carbon atoms are colored gray80. Non-carbon atoms of ligands are depicted in sphere with the size from big to small as: sulfur, oxygen, nitrogen and boron atoms. Water molecules are also depicted as spheres.
- 50 -
- 51 - Figure 2.3. Stereo view of interactions of the bound ligands within SHV-1 β-lactamase active site. (A) ceftazidime-BATSI; (B) cefoperazone-BATSI; (C) compound 1; (D) compound 2. Dashed black lines indicated hydrogen bonds.
- 52 -
- 53 - Figure 2.4. Comparisons of β-lactamases and their bound BATSIs. (A) All Cα superposition of ceftazidime-BATSI-SHV-1(magenta) and cefoperazone-BATSI -SHV-1
(orange) structures with wt SHV-1 (pale blue, PDB ID: 1SHV). The BATSI ligands and water molecules have been omitted for simplicity. Major shifts of active site residues have been indicated via black arrows. (B) Active site superposition of the structure of
SHV-1 β-lactamase in complex with ceftazidime-BATSI (magenta) with the ceftazidime-
BATSI complexed structures of TEM-1(cyan, PDB ID: 1M40), CTX-M (green, PDB ID:
1YLY) and AmpC (pale green, PDB ID: 1IEM) structures. The 11 Cα atoms used in superposition are residues 69, 70, 73, 105, 130, 132, 166, 170, 234, 236 and 237 of TEM-
1 and CTX-M, respectively on the equivalent residues of SHV-1/ceftazidime-BATSI
(superposition resulted in a r.m.s.d of 0.299Å and 0.333Å). The 12 Cα atoms used in the superposition are 63-67, 150-152, 315-318 of AmpC on SHV-1 residues 69-73, 130-132,
234-237 with a r.m.s.d of 1.338Å. (C) 11-active-site-residue superposition of the ceftazidime-BATSI-SHV-1 structure with the ceftazidime-BATSI-TEM-1 (cyan, PDB
ID: 1M40) structure. (D) 11-active-site-residue superposition of the ceftazidime-BATSI-
SHV-1 structure with the CTX-M (green, PDB ID: 1YLY) structure. (E) Active site of the structures of ceftazidimi-BATSI complexed SHV-1 (magenta), TEM-1(cyan, PDB
ID: 1M40) and CTX-M (green, PDB ID: 1YLY) β-lactamase. Ceftazidime-BATSI and water molecules have been omitted for clarity. All Cα superposition of TEM-1 and CTX-
M respectively against all Cα atoms of SHV-1- ceftazidime-BATSI resulted in a r.m.s.d of 1.598 and 2.706 respectively. The major movements in the active site have been indicated via black arrows. (F) Superposition of the ceftazidime-BATSI-SHV-1 structure
(magenta) with the structures of cefoperazone-BATSI-SHV-1 (orange), chiral-penicillin-
- 54 - BATSI-TEM-1(yellow, PDB ID: 1ERO) and chiral-cephalothin-BATSI-CTXM-9 (lemon color, PDB ID: 1YM1). (G) All Cα superposition of wt SHV-1 (pale blue, PDB ID:
1SHV), compound 1-SHV-1 (brown) and compound 2-SHV-1 (warm pink) with cefoperazone-BATSI -SHV-1 (orange). The superposition resulted in a r.m.s.d of 0.566,
0.407 and 0.441.
- 55 - Table 2.1. Inhibition data for BATSI compounds
Ki (µM)
ceftazidime-BATSI
SHV-1 2.2 ± 0.2(Thomson et al., 2007b)
TEM-1 M182T 0.39 (Caselli et al., 2001)
CTXM-9 0.015 (Chen et al., 2005)
AmpC 0.02 (Caselli et al., 2001)
ADC 0.31 ± 0.03
cefoperazone-BATSI
SHV-1 17 ± 2
ADC 0.60 ± 0.06
Compound 1
SHV-1 11.3 ± 0.6
Compound 2
SHV-1 9.8 ± 0.5
- 56 - Table 2.2. Data collection and refinement statistics
SHV-1 complexes Ceftazidime- Cefoperazone- compound 1 compound 2 BATSI BATSI Data collection space group P212121 P212121 P212121 P212121 cell dimensions 49.55, 56.48, 49.22, 56.14, 49.59, 55.64, 49.70, 55.48, a, b, c (Å) 82.07 82.59 83.04 83.16 wavelength (Å) 0.9184 1.0809 0.97946 0.97946 resolution (Å) a 50.00-1.75 50.00-1.33 50.00-1.30 50.00-1.24 (1.81-1.75) (1.38-1.33) (1.35-1.30) (1.28-1.24) Rsym 11.1 (47.4) 3.4 (22.9) 5.5 (32.9) 4.8 (24.0) I/σI 10.6 (2.8) 35.6 (3.9) 19.1 (3.1) 19.7 (3.8) Completeness (%) 98.8 (99.4) 93.5 (73.5) 96.9 (92.0) 98.3 (95.8) Redundancy 4.2 (4.1) 4.4 (2.9) 3.6 (3.6) 2.8 (2.8) Refinement Resolution range 33.92-1.75 24.03-1.33 24.78-1.30 24.21-1.24 (Å) (1.795- (1.364-1.330) (1.333-1.300) (1.273-1.241) 1.750) no. of reflections 22409 47303 52640 61493 Rwork/Rfree 15.32/18.11 17.90/19.01 18.56/20.31 17.07/19.37 no. of atoms: 2056, 22, 2087, 28, 183 2048, 30, 266 2046, 29, 278 protein, ligand, 219 water rmsd b bond length (Å) 0.012 0.007 0.011 0.010 bond angles 1.564 1.171 1.481 1.431 (deg) average B-factors (Å2) protein 9.334 11.119 14.782 11.957 active site 17.020 14.674 29.033 26.607 BATSI water 25.810 22.218 28.366 26.815 Ramanchandran plot statistics (%) core regions 91.3 92.6 91.3 90.5 additional 8.7 7.4 8.2 9.5 allowed generously 0.0 0.0 0.4 0.0 allowed disallowed 0.0 0.0 0.0 0.0 regions a Numbers in parentheses refer to the highest resolution shell. b rmsd, root-mean-square deviation
- 57 - CHAPTER 3
Crystal Structures of SHV-1 β-Lactamase in Complex with penem
and penam sulfone inhibitors that form cyclic inhibitory intermediates
3.1 Introduction
This chapter is focused on structural explorations of novel penem and penem sulfone β-lactamase inhibitors bearing heterocycle substitutions at the 6 position via a methylidene linkage (Figure 3.1). These two classes of compounds have broad inhibition spectrum effective against Class A, C and/or D β-lactamases (Bethel et al., 2008;Buynak et al., 1999;Pattanaik et al., 2009;Mansour et al., 2007). In this study, we selected penem
1 and SA1-204, being amongst the most potent inhibitors from the penem and penem sulfone inhibitor classes respectively, to study their mechanism of inhibition against a
Class A β-lactamase, SHV-1.
Wyeth developed a series of the 6-methylidene penem inhibitors, including monocyclic, [5,5]-bicyclic, [6,5]-bicyclic, and [5,5,5]-tricyclic heterocycles substitutions.
Their mechanism of inhibition was studied by kinetic, computational, and X-ray crystallographic methods using a typical monocyclic (penem 4), [6,5]-bicyclic (penem 2), and [5,5,5]-tricyclic (penem 3) substituted compounds (Mansour et al., 2007). The compounds first forms an acyl-intermediate with catalytic S70 concomitant with opening of the β-lactam ring. Subsequently, a remarkable 7-endo (sometimes 6-endo) trig rearrangement reaction leads to a 1,4-dihydrothiazepine acyl-enzyme complex (Figure
3.1A). However, the stereochemistry at the C7 position of the final complex displays
- 58 - diversity with both R and S C7 configurations observed. Elucidation of the absolute C7 configuration would help identify and predict favorable enzyme-inhibitor interactions to allow future design of more potent inhibitors. [5,5]-bicyclic penem 1 was chosen for this purpose in this study. Penem 1 has an IC50 value of 1 ± 0.4 nM against Class A TEM-1 and 1 ± 0.5 nM against Class C AmpC (Mansour et al., 2007) and a Ki value of 45 ± 8 nM against Class D OXA-1 β-lactamase (Bethel et al., 2008). A previous computational study predicted that this compound would form the C7 S configuratioin with Class A
SHV-1 β-lactamase and C7 R configuration with Class C GC1 β-lactamase. To probe these computational predictions, we trapped the penem 1 complex in SHV-1 β-lactamase crystals.
The 6-methylidene penam sulfone series were developed by the Buynak group
(Buynak et al., 1999) showing strong inhibitory properties against Class A and Class C β- lactamase. LN1-255 and SA1-204 both have C6 pyridylmethylidene; yet LN1-255 contains catechol features of the dihydrophenyl ring to improve the bacteria entry. SA1-
204 lacks this latter feature but has a better IC50 value. The relative IC50 values are 0.001,
0.04 and 0.39 µM of SA1-204 against P99, TEM-1 and PC1β-lactamase respectively; while these values are 0.026, 0.06 and 0.7 µM for LN1-255 against P99, TEM-1 and
PC1β-lactamase respectively (Buynak et al., 1999). LN1-255 was previous studied by mass spectroscopy and X-ray crystallography. Its proposed mechanism involves enzyme acylation followed by sequentially ring openings (Figure 3.1B). Subsequently, a cyclization reaction of LN1-255 results in a bicyclic acyl-enzyme complex. In contrast, the similar compound SA1-204 had previously been observed to yield significant
- 59 - amounts of Michaelis-Menten complex formation (Kalp et al., 2007). In this study we probed the structure of the SA1-204: SHV-1 complex by X-ray crystallography and observed a stable acyl-enzyme complex.
3.2 Materials and Methods
3.2.1 Enzyme purification
SHV-1 β-lactamase was expressed and purified as described previously (Padayatti et al., 2006;Ke et al., 2011).
3.2.2 Crystallization and soaking
SHV-1 β-lactamase was crystallized as described previously (Kuzin et al., 1999).
Briefly, a 5μl drop containing 2mg/ml SHV-1 β-lactamase and 0.56mM Cymal-6
(Hampton Research) in reservoir solution (20-30% PEG6000 in 100mM HEPES pH7.0) was equilibrated against 1ml reservoir solution. Crystals grew to full length in 2-3 days.
To obtain the SHV-1: penem 1 complex, crystals were soaked in mother liquor containing 40mM penem 1 overnight (21 hours). To obtain the SHV-1:SA1-204 complex, crystals were soaked in mother liquor containing 50mM SA1-204 for 90 minutes. After soaking, the crystals were cryo-protected with 20-25% 2-methyl-2, 4- pentanediol (MPD) in mother liquor containing the corresponding inhibitor and flash frozen in liquid nitrogen prior to data collection.
3.2.3 Data collection and structure determination
- 60 - X-ray diffraction data for the SHV-1: penem 1 complex was collected using in house Rigaku MicroMax-007 HF microfocus X-ray generator with Saturn 944+ CCD X- ray detector. This SHV-1: penem 1 dataset was collected of 360° rotation with a 0.5° image step at a single wavelength (Cu Kα wavelength of 1.542Å). The data were scaled with both anomalous reflections kept separate and as well by merging them. The anomalous scaled dataset was subsequently used to generate the anomalous difference
Fourier map using the FFT program (Ten Eyck, 2011) of CCP4 suite to identify the positions of sulfur atoms of penem 1 intermediate. X-ray diffraction data for the SHV-
1:SA1-204 complex was collected at the Brookhaven National Synchrotron Light Source beamline X-29.
Both data were processed using HKL2000 (Otwinowski, Minor, 1997). Structures were determined using chain A of the isomorphous crystal structure of SHV-1 β- lactamase (PDB 1VM1) (Kuzin et al., 2001). Refinement was carried out using
REFMAC (Murshudov et al., 1997) and model building was done using COOT (Emsley,
Cowtan, 2004). After initial refinement, density in the active site revealed an intermediate covalently attached to Ser70 residue. The PRODRG2 server (Schuttelkopf, van Aalten,
2004a) was used to obtain the parameter and topology files for the covalently attached inhibitor intermediates. Crystallographic refinement was monitored using the program
DDQ (van den Akker, Hol, 1999b) and the final model quality was assessed using
PROCHECK (Laskowski et al., 2001). Data collection and refinement statistics are summarized in Table 3.2.
- 61 - 3.3 Results and Discussion
3.3.1 SHV-1: penem 1 structure
The initial unbiased Fo-Fc map reveals a large unmodelled electron density emanating from catalytic Ser70 residue (Figure 3.2A left). Based on the shape of the density and the proposed reaction mechanism (Figure 3.1A), a 7-membered-ring acyl- intermediate was modeled into the density. There are three tautomers possible
(intermediate 7-9), which were all consider yet the density indicates that intermediate 8 is the dominant intermediate formed. Moreover, the anomalous signals of S atoms further confirmed positions of the two S atoms of the modeled species 8 (Figure 3.2A right).
Active site water molecules were examined carefully and included in refinement including the deacylation water near residues E166 and N170. The final R/Rfree is
18.7/23.4 and there are no residues are in the disallowed region of Ramanchandran plot
(Table 3.1).
Penem 1 forms a very stable acyl-intermediate in the enzyme active site. This acyl intermediate is also suggested by the mass spectroscopy (Thomson et al., 2007a). We collected a number of datasets with soaking time points ranging from 7 minutes to 21 hours all reveal the same single R-configuration at the stereo center C7 of the acyl- intermediate (data not shown). This intermediate has relatively few direct hydrogen bonding interactions with enzyme active site although there are ample van der Waals interactions (Figure 3.3A). As usual, the β-lactam carbonyl group is positioned in the oxyanion hole by forming strong H-bonds with backbone nitrogen atoms of S70 and
- 62 - A237. In addition, the NH-thiazepine atom is hydrogen bonded to the carbonyl atom of
S130 (3.2Å). The C3 carboxylate group points outwards and has water mediated interactions with D104, S106 and N132. The heterocyclic substitution lies above the polar side chain of N170 and also points toward the bulk solvent.
The overall complexed structure is similar to that of the apo SHV-1 structure with an rmsd 0.333Å of all Cα atoms in the superposition. However, the presence of the covalently bound penem inhibitor induced several changes in the enzyme active site. The most prominent change is the outward shift of the loop containing Y105, which releases the steric hindrance between the C3 carboxylate group and the Y105 containing loop
(Figure 3.4A). Compared with earlier crystallographic studies of similar penem inhibitors
(Nukaga et al., 2003a;Venkatesan et al., 2004), the positions of the two ring systems of the covalently bound inhibitors vary greatly (Figure 3.4A). The orientation of penem 1 product in the SHV-1 active site resembles that of the penem-products in GC1 active site.
However, compared to the previous SHV-1: penem complexes, all three acyl- intermediates adopt the R configuration but have two orientations yet differ from each other by an 180° rotation around the bond to the serine ester. However, penems 1, 2 and 3 have close IC50 values against TEM-1 and AmpC β-lactamases (Weiss et al., 2004).
Therefore, it is likely that the precise positioning of the inhibitor ring moieties within the active site after acylation is not a critical step in the inhibition process.
Interestingly, earlier computational study predicted that penem 1 would form a dihydrothiazepine acyl-intermediate with C7 S configuration and interact with SHV-1
- 63 - through π-π stacking and van der Waals interactions. Taken together, we could conclude that firstly, the hydrogen bonding interactions are the dominant interactions in the SHV-1 active site. For example, the carbonyl oxygen prefers to sitting in the oxyanion hole.
Secondly, it is potentially favorable to have Y105 in a less rigid position as also seen in the complex structures of SHV-1: boronic acid transition state inhibitor analogues (Ke et al., 2011). Thirdly, the size of the C7 heterocycle substitution affects the binding mode in the enzyme active site. Finally, the stability of the intermediate is not due to the low occupancy or disorder of the deacylation water molecule as proposed before (Nukaga et al., 2003a) since in our structure, the deacylation water is well refined as occupancy of
1.0. It is likely that the stability of the penem 1 intermediate is due to the decreased electrophilicity of the carbonyl carbon as a result of the conjugation of the acyl ester with the large dihydrothiazipine ring as was also pointed out previously (Venkatesan et al.,
2004). It is also interesting to note that the penem 1 is situated in close proximity to the
HEPES buffer molecule (Figure 3.3A). HEPES was used in both this study’s crystallization protocol as well in the previous SHV-1 crystallization protocols to obtain the previous penem complexes (Nukaga et al 2003). The proximity of the sulfone moiety of HEPES could perhaps be used to design novel penem inhibitors, similar to how the position of HEPES was used to rationally design the penam sulfone inhibitor SA2-13
(Padayatti et al., 2006).
3.2.2 SHV-1:SA1-204 structure
The initial unbiased omit Fo-Fc map reveals an unambiguous covalent acyl intermediate attached to the catalytic Ser70 residues with a characteristic feature of a
- 64 - bicyclic ring and a phenol tail (Figure 3.2B). Based on the proposed reaction mechanism
(Figure 3.1), a bicyclic acyl intermediate was modeled, which fits well with the density.
Its inclusion in refinement decreased the the R and Rfree indicating the correct placement of this intermediate. In addition, 261 water molecules were added to the model. As usual for most SHV-1 structures, one cymal-6 and one fragment of cymal-6 were included in refinement. At final stage, the mode has R/Rfree of 16.8/19.4 and no residues are in the disallowed region of Ramanchandran plot (Table 3.1).
In the active site of the SHV-1 SA1-204 complex, the carbonyl oxygen of SA1-
204 is positioned out of the oxyanion hole and stabilized by side chains of S130 and
K234 (Figure 3.3B). The bicyclic ring partially occludes the oxyanion hole and makes van der Waals interactions with A237. The C3-carboxylate group is stabilized by R244.
The C4-nitrogen and the sulfone group of SA1-204 interact with water molecules including a water-mediated interaction with Y105. The phenol tail is close to a cymal-6 molecule and in van der Waals distance with A217 and L220. The C2-methyl group is in van der Waals distance with V216.
The SHV-1:SA1-204 protein structure has a similar conformation as that of the structures of apo SHV and SHV-1:LN1-255 complex with rmsd of 0.357 and 0.109 respectively with all Cα superpositioning. Compared with apo SHV-1 structure, the most prominent movements of the active site residues are the different rotamer taken by
Ser130 and the outward shift of V216 containing loop to accommodate the carbonyl oxygen and the C2-methyl group of SA1-204 intermediate (Figure 3.4B). The
- 65 - reorientation of the ester carbonyl away from the oxyanion hole and pointing toward
S130 has previously also been observed and likely accounts for the slow deacylation rate of imipenem against TEM-1β-lactamase and LN1-255 against SHV-1 β-lactamase
(Pattanaik et al., 2009;Maveyraud et al., 1998). Additional reasons for the decreased deacylation rate and stability of the SA1-204 intermediate include the steric and electrostatic barrier and the spatially increased distance to the approach of the deacylation water to the ester carbonyl. This is also the case for SA1-204 against SHV-1 as seen from our structure (Figure 3.3B). First, the deacylation water is positioned 4.07Å away from the ester carbonyl compared while in the usual acyl intermediate this distance is 2.77Å.
Secondly, the bicyclic aromatic ring decreased the electrophilicity of the ester carbonyl due to the conjugating effect. In addition, the bulky bicyclic aromatic ring imposes steric hindrance to the approach of the deacylation water to the ester carbonyl.
SA1-204 is very similar to LN1-255 differing only by two hydroxyl moieities.
Comparison of these two penam sulfones indicates that SA1-204 is more potent than
LN1-255 by comparing the IC50 values against three representative serine β-lactamases
(Buynak et al., 1999). The IC50 values are 0.001, 0.04 and 0.39 µM of SA1-204 against
P99, TEM-1 and PC1β-lactamase respectively; while these values are 0.026, 0.06 and 0.7
µM of LN1-255 against P99, TEM-1 and PC1β-lactamase respectively (Buynak et al.,
1999). And this is consistent with the structural observations. SA1-204 is an analogue of
LN1-255 and contains a C2-phenylacetate substitution instead of a C2-catecholicacetate group (Figure 3.1B). This C2 substitution of LN1-255 has two alternative conformations when complexed with SHV-1 while SA1-204 has only one conformation in the SHV-1
- 66 - active site (Figure 3.4B). Based on previous Raman studies, the greater inhibition efficacy of SA1-204 was ascribed to its long time blocking of the enzyme active site as the unreacted Henri-Michaelis complex of up to one hour (Kalp et al., 2007). However, we did not capture this Henri-Michaelis complex. We had structures at different timepoints (10 min, 50 min, 60 min and 90 min, data not shown) which all showed the same unambiguous bicyclic aromatic acyl complex. A potential reason is that this inhibitor has a hydrophobic tail that is very similar to cymal-6 (Figure 3.1B), therefore, some of the excess SA1-204 might bind with its hydrophobic tail first where cymal-6 binds during the Raman experiments since cymal-6 was not included in the Raman soaking studies. In our soaking studies, we do include cymal-6 so the inhibitor then likely goes only to the catalytic S70 residue for a reaction to form an acyl enzyme complex.
Alternatively, it could be that the Michaelis-Menten complexes claimed by Raman studies are actually the unreacted SA1-204 remaining in the buffer or solution because the IC50 value is extremely low in nM range. Based on our studies, the acyl-intermediate of SA1-204 is quite stable that once it is covalently attached to the active site it will relatively resistant to deacylation.
- 67 - A
- 68 - B
Figure 3.1. Chemical structures and reaction schemes. (A) Chemical structures of WAY compounds and the proposed inhibition mechanism by a generalized penem. (based on
Knox’s work and others) (Nukaga et al., 2003a;Venkatesan et al., 2004); carbon atoms labeled with * are the stereo centers; (B) Chemical structures of SA1-204, LN1-255 and
Cymal-6. The proposed reaction was taken from Kalp et al (Kalp et al., 2007).
- 69 - A
B
- 70 - Figure 3.2. Electron density maps are depicted (A) Left figure is the unbiased omit Fo-
Fc map contoured at 2.0σ of SHV-1: penem 1 complex; right figure is the anomalous difference Fourier map contoured at 3.5σ showing the electron densities right on top of the two sulfur atoms of penem 1 intermediate; (B) Unbiased omit Fo-Fc map contoured at 2.5σ of SHV-1:SA1-204 complex. Ligand carbon atoms are colored black. The color schemes are nitrogen atoms gray20, oxygen atoms gray40 and sulfur atom gray60. SHV-
1 β-lactamase carbon atoms are colored gray90. Non-carbon atoms of ligands are depicted in sphere with the size from big to small as: sulfur, oxygen, nitrogen and boron atoms. Water molecules are omitted for simplicity.
- 71 - A
B
Figure 3.3. Stereo view of interactions of the bound ligands within SHV-1 β-lactamase active site. (A) penem 1 reaction intermediate; (B) SA1-204 reaction intermediate.
Dashed black lines indicated hydrogen bonds. Water molecules are shown as spheres and labeled as numbers. Water molecules labeled as 54 in A and 22 in B are the deacylation waters. The color scheme is the same as in Figure 3.2.
- 72 - A
- 73 - B
Figure 3.4. Structural superpositions. (A) Superposition of SHV-1: penem 1 complex structure with apo SHV-1 structure and earlier penem complexed structures. The C7 S configuration of GC1:penem 3 is omitted for simplicity. The PDB IDs are 1SHV, 1ONG,
1ONH, 1Q2P and 1Q2Q repectively. SHV-1: penem 1 is colored black; apo SHV-1 and earlier penem complexed structures are colored grey. Deacylation water molecules are depicted as spheres with arrow-pointing and colored correspondingly. (B) Superposition of SHV-1: SA1-204 complex structure with apo SHV-1 structure and SHV-1: LN1-255 complex structure. SHV-1:SA1-204 is colored black; SHV-1:LN1-255 and apo SHV-
1(PDB ID: 1SHV) are colored grey. SA1-204 is depicted as ball and stick representation
- 74 - Table 3.1. Data collection and refinement statistics SHV-1 : penem 1 SHV-1 : SA1-204 Data collection
space group P212121 P212121 cell dimensions a, b, c (Å) 49.58 55.50 85.80 49.64 55.58 83.35 α, β, γ (deg) 90.00 90.00 90.00 90.00 90.00 90.00
wavelength (Å) 1.54178 1.0810 resolution (Å) 50.00-1.84 (1.91-1.84) 50.00-1.53 (1.58-1.53) Rsym 7.2 (17.3) 9.2 (29.1) I/σI 26.6 (8.1) 39.4 (3.8) Completeness (%) 98.2 (90.7) 97.8 (84.9) Redundancy 11.9 (9.8) 6.3 (4.7) Refinement Resolution range (Å) 20.01-1.84 (1.885-1.837) 27.68-1.53 (1.573-1.533) no. of reflections 19909 32972 Rwork/Rfree 18.7/23.4 (25.4/37.1) 16.8/19.4 (23.4/31.6) no. of atoms: protein, inhibitor, water, others 2037, 20, 243, 60 2046, 32, 261, 44 b rmsd bond length (Å) 0.006 0.009 bond angles (deg) 1.272 1.306 average B-factors (Å2) protein, inhibitor, water, others 14.4, 18.9, 29.9, 26.5 16.3, 19.0, 31.1, 26..3 Ramanchandran plot statistics (%) core regions 93.5 90.9 additional allowed regions 6.5 8.7 generously allowed regions 0.0 0.4
disallowed regions 0.0 0.0 a Numbers in parentheses refer to the highest resolution shell. b rmsd, root-mean-square deviation
- 75 - CHAPTER 4
Trans-Enamine Intermediate Formation as a β-Lactamase Inhibition Strategy
Probed for Inhibitor-Resistant S130G SHV β-Lactamase
4.1 Introduction
Inhibitor resistant (IR) β-lactamases threaten the current antibiotic armamentarium by overcoming the effectiveness of the β-lactamase inhibitors, leading to resistance to the current set of clinical β-lactam/β-lactamase inhibitor combinations(Drawz, Bonomo, 2010). New inhibitors are currently being developed with the aim of overcoming these resistance phenotypes. Our novel SA2-13 inhibitor is one such compound (Padayatti et al., 2006). To date, clinically observed inhibitor resistant variants in the Class A SHV and TEM β-lactamases are found to have amino acid substitutions at Ambler positions 69, 130, 234, 244, 275, and 276
(www.lahey.org/studies/webt.asp) (Drawz, Bonomo, 2010). In this study we are focusing on S130G SHV to study the mechanism of IR by protein x-ray crystallography.
The S130G mutation occurs in the highly conserved SDN loop of Class A β- lactamases. It was suggested that in Class A β-lactamases, S130 participates in substrate recognition, facilitates β-lactam ring opening during enzyme acylation, and initiates the irreversible step of inactivation by covalently cross-linking to S70 residues (Figure 4.1)
(Kuzin et al., 2001;Imtiaz et al., 1994;Imtiaz et al., 1993;Atanasov et al., 2000;Vakulenko et al., 1998;Lamotte-Brasseur et al., 1991). To elucidate the mechanism of inhibitor resitance caused by the S130G mutation, numerous studies have been carried out
- 76 - including molecular modelling, kinetics, protein crystallograpy, mass spectrometry and
Raman crystallography techniques (Helfand et al., 2003;Helfand et al., 2007), (Thomas et al., 2005), (Sun et al., 2004). Kinetic experiments suggested that S130G SHV is resistant to clavulanic acid but not tazobactam. Molecular modelling and thermal denaturation indicated that both S130G SHV and S130G TEM are stable compared to the corresponding apo enzyme. Molecular modelling also predicted that a water molecule can compensate the loss of the hydroxyl group in the S130G mutant which was later demonstrated by X-ray crystallographic study and explained the reason for the presence of the residual hydrolytic activity of S130G mutant. Additionally, protein crystallograpy captured two species in the active site of S130G SHV reacted with tazobactam. The primary one is the cis-enamine intermediate or enamine species with the ester carbonyl group primed out of the enzyme’s oxyanion hole. And the other one is the aldehyde product (Sun et al., 2004). Mass spectrometry studies revealed that S130G SHV is inhibited by tazobactam through formation of stable covalently bound products: an aldehyde and a hydrated aldehyde (Pagan-Rodriguez et al., 2004). Mass spectrometry studies also revealed that S130G SHV is inhibited by clavulanic acid through formation of a stable aldehyde covalently attached to S70 (Sulton et al., 2005). Moreover, Raman crystallography observed the formation of trans-enamine intermediates of S130G SHV with tazobactam, clavulanic acid and SA2-13 (Helfand et al., 2007). SA2-13 is a rationally designed inhibitor by our group based on the strategy of trapping and stablilizing the inhibitor into trans-enamine intermediate to form suicide inhibitors. SA2-
13 was demonstrated to have a ~10-fold improvement in stabilization of this key inhibitory intermediate in SHV-1 β-lactamase (Padayatti et al., 2006). To further test this
- 77 - strategy against IR β-lactamases, we determined the crystal structure of SA2-13 in complex with IR S130G SHV.
4.2 Materials and Methods
4.2.1 Inhibitors
SA2-13 was synthesized as previously described (Padayatti et al., 2006).
4.2.2 Mutagenesis, purification and crystallization.
S130G mutant of SHV-1 β-lactamase was generated by site-directed mutagenesis using Stratagene’s Quick Change Mutagenesis Kit (Helfand et al., 2003;Hujer et al.,
2001). S130G SHV was expressed, purified and crystallized as previously described
(Helfand et al., 2007;Hujer et al., 2001;Kuzin et al., 1999). This crystal was provided by
Dr. Helfand.
4.2.3 Soaking, data collection and structure determination
S130G SHV β-lactamase crystals were soaked with mother liquor containing
50mM SA2-13 for 30 minutes. Then the soaked crystal was transferred to the cryprotectant for 1 minute before being flash frozen in liquid nitrogen. The cryoprotectant is 25% MPD in mother liquor containing the corresponding inhibitor. X- ray diffraction data was collected at the Advanced Light Source (ALS) synchrotron
(beamline 4.2.2). Data were processed using d*TREK (Pflugrath, 1999). Structures were determined by molecular replacement using program Phaser (McCoy et al., 2007b) with
- 78 - the search model of uncomplexed SHV-1 structure (PDBID: 1VM1; tazobactam and waters were removed). S130 was changed to Glycine in COOT prior to subsequent refinement (Emsley, Cowtan, 2004). Refinement was carried out using REFMAC
(Murshudov et al., 1997) and model building was done using COOT (Emsley, Cowtan,
2004). After initial refinement, strong density in the active site was found to extend from the hydroxyl group of S70, suggesting an intermediate covalently attached to S70 residue.
We first had Cymol-6 and waters included in the refinement. Then we carefully placed the trans-enamine form of SA2-13 into the difference density. The PRODRG2 server was used to obtain the parameter and topology files for the modeled intermediate.
Crystallographic refinement was monitored using the program DDQ (van den Akker,
Hol, 1999c)and the final model quality was assessed using PROCHECK (Laskowski et al., 2001). Data collection and refinement statistics are shown in Table 1.
4.3 Results
4.3.1 S130G:SA2-13 Complex.
The crystal structure of SA2-13 bound to S130G SHV was determined to 1.45Å resolution. The unbiased omit |Fo|-|Fc| map revealed a well ordered intermediate covalently attached to Oγ of Ser70 residue (Figure 4.2). The overall structure resembled structures of apo SHV-1 (PDB ID: 1SHV) and apo S130G SHV (PDB ID: 1TDL) with an all Cα superposition rmsd of 0.350 and 0.136 respectively. As seen in the apo S130G
SHV structure, the active site of our structure has a mutated Gly residue at position 130 and an expanded binding site with an increased Cα-Cα distance between G130 and S70.
The trans-enamine intermediate was modeled because the torsion angle of C7-C6=C5-N4
- 79 - was refined to 154.47°. The inclusion of the trans-enamine intermediate brought down the R/Rfree considerably. The final R-factor is 18.05% and Rfree is 19.71%. Throughout the refinement, the occupancy of the modeled intermediate was kept at 1.0.
4.3.2 Interactions of the intermediate in the active site.
Functional groups of SA2-13 such as the sulfone, both carboxyl groups and the ester carbonyl are well-ordered as revealed by the electron density map (Figure 4.2). The ester carbonyl resides in the oxyanion hole hydrogen bonded to backbone nitrogen atoms of S70 and A237. The C3 carboxylate is stabilized by N170 and N132. The sulfone group interacts with 3 water molecules. The carboxylate group makes salt bridges with K73 and
K234 and has a water mediated hydrogen bond with the carbonyl oxygen of C16 (Figure
4.3).
4.4 Discussion
As predicted by Raman crystallography (Helfand et al., 2007), SA2-13 forms a well stabilized trans-enamine intermediate with the IR S130G SHV. SA2-13 is a very adaptable inhibitor against both WT SHV-1 and IR S130G SHV. When comparing the
SA2-13:S130G complex to the SA2-13: WT SHV-1 complex (PDB ID: 2H5S), a negligible all Cα superposition RMSD of 0.15Å is observed. In addition, SA2-13 is bound in the active site of S130G SHV very much like in the WT SHV-1 structure except for the reposition of the carboxyl addition (Figure 4.4A). In both structures, intermediates are refined to be a trans-enamine species and the positions of the ester carbonyl, the C3 carboxylate and the sulfone are similar. With S130 mutated to Glycine, the carboxylate
- 80 - tail of SA2-13 now takes the position where normally the hydroxyl group of Ser130 is located and as a result is stabilized by an additional salt bridge with K73. In addition, the active site of S130G SHV has changed little (Figure 4.4A). The most significant differences are that S130 has mutated to Gl30 and that N170 only takes an outward conformation accompanied by the changed hydration pattern in the active site (Figure
4.4B). In the structure of SHV-1 SA2-13 complex, the deacylation water molecule takes two alternative positions (W1’ and W2’, 0.5 occupancy of each) corresponding to the two conformations of N170. In our structure, the deacylation water molecule (W1) has occupancy of 1.0 and positioned by E166 and W2 water mediated interaction with N170.
N170 takes only one conformation and interacts with the C3 carboxylate group of SA2-
13. This W2 water molecule was not observed in the structures of apo SHV-1 (PDB ID:
1SHV) and SHV-1-SA2-13 complex (PDB ID: 2H5S).
In conclusion, SA2-13 is a very adaptable inhibitor which could form stable trans-enamine species with both WT SHV-1 and S130G SHV. As a penam sulfone like tazobactam, the trans-enamine is stabilized by the intramolecular hydrogen bond between the sulfone moiety and the N4 of SA2-13. Additionally, the carboxyl-linker (R2 group) sticking into the carboxylate binding cavity further stabilized the acyl-enzyme trans- enamine species and therefore likely prevented it to tautomerize back to the imine intermediate. However, SA2-13 has poor penetration into bacteria (personal communication with Dr. Bonomo). Therefore for future inhibitor improvement, modifications of SA2-13 are needed so that it could get into the cell. For example, an amide-linker (R2 group) could be tested, which is more hydrophobic than that of the carboxyl-linker.
- 81 - A
B
Figure 4.1. (A) Chemical structure of Clavulanic acid, tazobactam and SA2-13; (B)
Proposed reaction scheme of a generalized inhibitor with Class A β-lactamases based on previous work (Sun et al., 2004).
- 82 -
Figure 4.2. Electron density of the SA2-13 compound in the active site of S130G SHV-1
β-lactamase. Unbiased omit Fo-Fc map is contoured at 2.5σ. SA2-13 carbon atoms are colored black. The color schemes are nitrogen atoms gray20, oxygen atoms gray40, and sulfur atom gray60. SHV-1 β lactamase carbon atoms are colored gray80. Non-carbon atoms of ligands are depicted in sphere with the size from big to small as: sulfur, oxygen, and nitrogen atoms. Water molecules are omitted for simplicity.
- 83 -
Figure 4.3. Stereo view of interactions of SA2-13 within S130G SHV β-lactamase active site. Dashed black lines indicated hydrogen bonds. The color scheme is the same as in
Figure 4.2.
- 84 - A
B
- 85 - Figure 4.4. All Cα superposition of SA2-13-S130G SHV (black) with SA2-13-SHV-1
(gray80, PDB ID: 2H5S). (A) Only water molecules interacting with the carboxylate tail are shown for simplicity. Major shifts of SA2-13 have been indicated by black arrows.
(B) W1 and W2 in black color are water molecules in the active site of SA2-13-S130G;
W1’ and W2’ in gray color are water molecules in the active site of SA2-13- SHV-1.
Enzymes are shown as cartoon representation on the background. Active site residues 70,
73, 104, 105, 130, 132, 170, 234, 235,237 are shown as sticks. All Cα superposition resulted in an rmsd of 0.158. The color scheme of oxygen and nitrogen atoms is the same as in Figure 4.2. Dashed lines indicated hydrogen bonds within active sites of SHV-1-
SA2-13 (gray) and S130G SHV-1-SA2-13 (black).
- 86 - Table 4.1. Data collection and refinement statistics
SA2-13 : S130G SHV Data collection
space group P212121 cell dimensions: a, b, c (Å) 49.67, 55.48, 83.62 α, β, γ (deg) 90.00, 90.00, 90.00 wavelength (Å) 0.9790 resolution (Å) 46.23-1.45 (1.50-1.45) Rsym 5.5 (37.9) I/σI 9.9 (2.0) Completeness (%) 99.3 (97.5) Redundancy 3.17 (2.35)
Refinement Resolution range (Å) 33.84-1.45 (1.488-1.450) no. of reflections 39329 Rwork/Rfree 18.05/19.71 no. of atoms: protein, SA2, water 2112, 24, 248 b rmsd bond length (Å) 0.009 bond angles (deg) 1.311 average B-factors (Å2) Protein, SA2, water 12.914, 22.589, 28.321 Ramanchandran plot statistics (%) core regions 92.2 additional allowed regions 7.4 generously allowed regions 0.4 disallowed regions 0.0 a Numbers in parentheses refer to the highest resolution shell. b rmsd, root-mean-square deviation
- 87 - CHAPTER 5
Crystal Structure of KPC-2 β-Lactamase
Crystal structure of KPC-2: insights into carbapenemase activity
in Class A β-lactamases (Ke et al., 2007)
5.1 Introduction
Carbapenems (imipenem, meropenem, and ertapenem) are the “last resort” β- lactam antibiotics for treating serious infections caused by multi-drug resistant gram- negative bacteria. Unfortunately, the acquisition of carbapenem-hydrolyzing β- lactamases by bacteria has resulted in a major threat to the clinical utility of these compounds (Babic et al., 2006;Paterson, 2006). Currently the Klebsiella pneumoniae carbapenemases (KPC-type β-lactamases), are rapidly emerging as a major threat in the
New York area (Bratu et al., 2005b;Woodford et al., 2004;Bratu et al., 2005a),
Pennsylvania (Pope et al., 2006), and internationally (Naas et al., 2005;Navon-Venezia et al., 2006). Three KPC variants have been found so far (KPC-1,-2, and -3) of which KPC-
1 and KPC-2 are almost indistinguishable, whereas KPC-3 exhibits different hydrolytic properties (Alba et al., 2005).
KPC carbapenemases belong to the Ambler class A β-lactamases (E.C. 3.5.2.6)
(Yigit et al., 2001;Ambler et al., 1991). In common with other class A β-lactamase, these highly proficient class A enzymes have an efficient hydrolysis ‘machinery’ involving a catalytic serine residue (S70), which acylates β-lactam substrates, and a well-positioned deacylation water. S70 is involved in the nucleophilic attack at the carbonyl carbon of the
- 88 - substrate leading to cleavage of the bond with the β-lactam ring nitrogen for penicillin and other substrates (Figure 5.1). The deacylation water molecule is primed by E166 and
N170 for nucleophilic attack of the β-lactam-acyl-enzyme intermediate and serves to liberate the β-lactam substrate from the active site by deacylation. Both acylation and deacylation involve a nucleophilic attack which is facilitated by a correctly positioned oxyanion hole (formed by the backbone nitrogens of residues 70 and 237) that attracts the carbonyl oxygen atom. These described active site elements are supported by a complex hydrogen bonding network involving a pair of conserved lysines (K37 and K234), N132, and S130.
Serine carbapenemases such as KPCs are unique among the class A β-lactamases because of their ability to hydrolyze β-lactams containing a substituent at the α position of the carbon atom in the β-lactam ring adjacent to the carbonyl (Swaren et al.,
1998;Sougakoff et al., 2002). Examples of such readily hydrolyzed β-lactams by carbapenemases include imipenem (a carbapenem containing a 6α-hydroxy-ethyl moiety) but also cefoxitin (a cephamycin containing a 7α-O-CH3 group)(Figure 5.1) (Alba et al.,
2005;Swaren et al., 1998;Sougakoff et al., 2002). Previous crystallographic studies of the class A serine carbapenemase NMC-A and SME-1 did not yield a unified conclusion regarding their carbapenemase phenotype. Interpretation of the NMC-A structure suggested a role for the slightly shifted position of N132 to perhaps accommodate these
α-substituents (Mourey et al., 1998;Swaren et al., 1998) whereas the SME-1 structure suggested a needed conformational change for catalysis and importance for the C69-
C238 disulfide, being in close proximity of the active site (Sougakoff et al., 2002).
Mutagenesis studies of the C69-C238 residues in SME-1 confirmed the critical role of
- 89 - this disulfide bond, but its function was not limited to carbapenemase activity since its disruption caused loss of hydrolytic activity towards all β-lactam antibiotics (Majiduddin,
Palzkill, 2005). Based upon mutagenesis studies it was concluded that no single residue within the active site of SME-1 is solely responsible for carbapenemase activity and that this activity is likely due to multiple simultaneous substitutions and accompanying conformational rearrangements that prime the active site to accommodate and hydrolyze carbapenems and cephamycins (Majiduddin, Palzkill, 2005).
The unresolved structural basis of this carbapenemase activity seen in class A enzymes and the urgent clinical threat presented by KPC β-lactamases found in
Klebsiella, Enterobacter, and Salmonella spp. prompted our investigation of KPC-2. We present here the structure of KPC-2 determined to 1.85Å resolution. Our analysis of
KPC-2 in comparison to other class A β-lactamase enzymes reveals alterations in active site topology with respect to non-carbapenemases such as SHV-1 and TEM-1 that could rationalize the structural basis of carbapenemase activity. Furthermore, the active site of
KPC-2 contains a well-resolved bicine molecule with its carboxyl moiety occupying a space that is likely involved in recognizing the carboxyl moiety of β-lactams thus providing insights into substrate recognition.
5.2 Materials and Methods
5.2.1 Enzyme expression and purification
The KPC-2 β-lactamase is expressed using the vector pBR322-catI-blaKPC-2 in
E. coli DH10B cells (a kind gift of Dr. Fred Tenover, the Centers of Disease Control and
Prevention, Atlanta GA). The plasmid containing blaKPC-2 was described by Yigit et al
- 90 - (Yigit et al., 2003) and has been confirmed by DNA sequencing. Transfected cells were grown in 500 ml of Luria Bertaini (LB) broth containing chloramphenicol 20 ug/ml and grown for 18 hrs and pelleted by centrifugation (5000g x 15min). Cell pellets were resuspended in 20mM Tris-Cl, pH 7.0 and periplasmic proteins were released using stringent periplasmic fractionation with lysozyme and EDTA as previously described
(Hujer et al., 2001). The supernatant was filter sterilized, passed through phenylboronate column (MoBiTec), which has specific binding affinity for class A β-lactamases, and
KPC-2 was eluted using 0.5M boronate/0.5M NaCl, pH 7.0. The eluent was loaded on
Superdex 75 size exclusion column and fractions containing nitrocefin-hydrolyzing activity were pooled and concentrated to 13mg/ml in 10mM Tris-HCl pH 7.6 buffer
(protein concentration was measured using Bradford method). The homogeneity of KPC-
2 was estimated at ~99% as assessed by SDS-PAGE. Carbapenemase activity was confirmed by hydrolysis of imipenem (imipenem λ = 299; Δε = -9000 M-1cm-1).
5.2.2 Crystallization
Initial crystallization screens were carried out using the Grid PEG6000 Screen kit
(Hampton Research Inc.) resulting in small needle crystal clusters. Crystallization conditions were optimized and diffraction quality KPC-2 crystals could be obtained using the sitting drop method and 0.7μl protein solution was mixed with 0.3 μl reservoir solution and equilibrated against a reservoir solution containing 16% PEG6000 in 0.1M bicine, pH 9.0. Crystals grew to full size in 3-14 days.
5.2.3 Data collection and structure determination
- 91 - For crystallographic data collection, a single crystal was transferred to mother liquor containing 15% glycerol for 1 minute after which the crystal was flash frozen in liquid nitrogen. A 1.85Å resolution diffraction data set was collected at APS 19BM at
100K. The crystals belong to trigonal space group P31 with cell parameters a=116.245 Å, b=116.245 Å, c=52.001Å, α=90OC, β=90 OC, γ=120 OC. The diffraction data were integrated and scaled using HKL2000 (Otwinowski, Minor, 1997) (see Table 5.1 for processing statistics).
The KPC-2 structure was solved by molecular replacement using the program
PHASER (McCoy et al., 2005) with chain A of the SME-1 structure (PDB identifier
1DY6) (Sougakoff et al., 2002) as the initial search model. PHASER found 3 molecules in the asymmetric unit resulting in a solvent content of 47%. The top solution underwent several rounds of REFMAC (Murshudov et al., 1997) refinement and manual model building with COOT (Emsley, Cowtan, 2004) which resulted in an R/Rfree of 27.1/33.2%.
The inability to further improve the R-factors suggested a possible twinning problem which was confirmed using the Crystal Twinning Server (Yeates, 1997). Subsequent refinement was carried out using CNS (Brunger et al., 1998b) with twinning operation
"h,-h-k,-l" and twinning fraction 0.420. Simulated annealing refinement followed by temperature factor refinement and manual model building lowered the R-factors considerably. No non-crystallographic symmetry restraints were used in refinement due to the relatively high resolution of the diffraction data. Electron density was of excellent quality for most of the residues (see Figure 5.2A for representative density near one of the active sites). Throughout the refinement, electron density for a bicine buffer molecule
- 92 - in each of the three KPC-2 molecules was apparent (Figure 5.2B). Therefore, three bicine buffer molecules were included in refinement as well as 294 water molecules and the final model containing residues 30-292 for each molecule (see Table 5.1 for additional details). The progress of crystallographic refinement was monitored using the program
DDQ (van den Akker, Hol, 1999d) and the final model quality was assessed using
PROCHECK (Laskowski et al., 2001).
5.3 Results
The structures of three KPC-2 molecules present in the asymmetric unit are refined to an R/Rfree of 14.8/19.0% and 90% of the residues had phi-psi angles in the acceptable region of the Ramanchandran plot (Table 5.1 and Figure 5.3A). Each molecule contains one cis-peptide (residue 167) which is common amongst other class A
β-lactamase structures. The three refined KPC-2 molecules in the asymmetric unit are very similar to each other as reflected by the low r.m.s.d. of 0.29-0.34 Å for superposition of all Cα atoms (Figure 5.3B). The active site residues are also in very similar position as well. Since these three molecules are very similar, we will be limiting our discussion and analysis to KPC-2 subunit A.
The structure of KPC-2 contains two subdomains generating a cleft resulting in an overall fold that is expectedly similar to that of other class A β-lactamases (Figure 5.3A).
One of the subdomains is largely α-helical whereas the other subdomain contains a 5- stranded β-sheet flanked by α-helices. The cleft generated by these two subdomains harbors the active site containing the catalytic S70 residue and also harbors the deacylation water that is primed by interacting with E166 and N170 as well as S70
- 93 - (Figure 5.4). The oxyanion hole formed by the backbone nitrogens of S70 and T237 is partially occluded by the side chain of S70 which is somewhat unusual for class A β- lactamases as will be discussed later. Adjacent to S70 is residue C69 which is involved in a disulfide bond with C238. This disulfide bond is characteristic for class A carbapenemases including NMC-A and SME (position 69 in class A enzymes usually is a
M or other hydrophobic residue). Both conserved lysines (K73 and K234) are also present in the active site of KPC-2 as well as residue N132. The importance of these residues have been well established in class A β-lactamases (Minasov et al.,
2002;Matagne et al., 1998). The entrance of the active site harbors W105 and R220 situated on opposite sides of the active site (Figure 5.4).
The active site contains a serendipitous, though interesting, finding in the form of a bound bicine buffer molecule. A bicine molecule is observed in the active site of all three KPC-2 molecules present in the asymmetric unit. The density is clearest for bicine in KPC-2 molecule A, with an average B-factor for bicine of 25Å2, and its omit electron density is depicted in Figure 5.2B. The B-factors for bicine in molecules B and C refined to somewhat higher average temperature factors of 36 and 45 Å2, respectively. Bicine
(di(hydroxyethyl)glycine) is a zwitterionic compound and contains a tertiary amine, a carboxyl group and two hydroxyethyl groups (Figure 5.1). Its carboxyl moiety is involved in the majority of bicine’s interactions in the active site by making hydrogen bonds with T235, T237, S130, a salt-bridge interaction with K234, a more distant ~4Å electrostatic interaction with R220, and some water-mediated interactions (Figure 5.4).
The two hydroxyethyl moieties of bicine do however not make direct hydrogen bonds with the protein (only a water-mediated interaction involving Wat3) but do provide
- 94 - hydrophobic interactions with W105. In addition, W105 could also be involved in a possible cation-π interaction of bicine’s tertiary amine group (Figure 5.4). Preliminary kinetic analysis showed that bicine had some detectable though weak inhibitory effect on
KPC-2 activity (measured up to 900 mM) and we therefore estimate its affinity is likely around 0.1M range, close to the 100mM buffer concentration during the crystallization experiment.
5.4 Discussion
The structure of KPC-2 at 1.85 Å resolution provides a new understanding of the active site of a clinically important carbapenemase responsible for the widespread emergence of imipenem-resistance in K. pneumoniae and other bacteria. Thus, the details revealed by our study are best appreciated in light of the structures of other serine class A β-lactamases. The precise mechanism that accounted for the carbapenemase phenotype in the class A β-lactamases, NMC-A and SME-1, was not precisely defined.
The atomic structures of NMC-A and SME-1 when compared to TEM did not reveal a unifying structural basis for this enhanced catalytic activity (Swaren et al.,
1998;Sougakoff et al., 2002). Nevertheless, the carbapenemase vs. non-carbapenemase hydrolytic differences are dramatic since the former can hydrolyze carbapenems with
-1 -1 great efficiency: the kcat/Km for imipenem hydrolysis is only 2.4 mM s for TEM-1
(Raquet et al., 1997), non-detectable for SHV-1 (Poirel et al., 2003), yet is 100-4,500 fold
- more efficient for carbapenemases with kcat/Km values of 300, 440, 1900, and 11300 mM
1s-1 for KPC-2, SME-1, KPC-3, and NMC-A, respectively (Alba et al., 2005;Swaren et al., 1998;Sougakoff et al., 2002). A similar trend is observed for the cephamycin
- 95 - cefoxitin which is also hydrolyzed more efficiently by the carbapenemases, compared to
TEM-1 and SHV-1 (Neu, 1983;Alba et al., 2005;Swaren et al., 1998;Sougakoff et al.,
2002). Compared to these Class A structures, the KPC-2 structure offers key insights into this novel kinetic behavior.
Superposition of the Cα atoms of KPC-2 onto NMC-A, SME-1, SHV-1, and
TEM-1 results in r.m.s.d. values of 0.81Å, 0.80Å, 1.38Å, 1.20Å, respectively (for 256,
256, 235, and 240 superpositioned Cα atoms). Hence, KPC-2, SME-1, and NMC-A are quite similar in overall structure (Figure 5.5) with the largest deviations located distant from the active site (near loop residues 88 and 98, Figure 5.5A). The sequence conservation shows a similar trend as KPC-2 is 52% and 54% sequence identical with
SME-1 and NMC-A, yet only shares 38% and 36% identity with SHV-1 and TEM-1, respectively. Most of the active site residues are remarkably conserved between class A carbapenemases and non-carbapenemases except for a few residues that are different in the latter (Figure 5.5B). These residues are T237 (is similar S in NMC-A and SME-1 yet is A in SHV-1 and TEM-1), H274 (M in SHV-1 and TEM-1), R220 (although an R is present in SHV-1 and TEM-1 it comes from residue R244 instead), and T216 (M in
SHV-1 and TEM-1). T237, H274, and R220 are all near the carboxyl moiety of bicine suggesting an important role for these residues in that region of the active site, perhaps also interacting with the carboxyl moiety of the carbapenem and cephamycin substrates.
Buried behind the active site is also the disulfide bond formed by C69 and C238 which is not present in SHV-1 and TEM-1. In addition to these residue differences, the active sites of the three carbapenemase structures reveal a number of conserved shifts with respect to
- 96 - the non-carbapenemases, SHV-1 and TEM-1 β-lactamases (Figure 5.5B, Table 5.2 and
3), suggesting a functional significance. These shifts are significant since they are all larger than the 0.2Å ESD coordinate errors reported for these structures (coordinate errors are listed in Table 5.2).
Comparing SHV-1 and TEM-1 vs. KPC-2, NMC-A, and SME-1, we observed that the following active site alterations are unique amongst the carbapenemases. Firstly, the decreased length of the pocket containing two water molecules Wat1 (deacylation water) and Wat2 (occupies temporarily oxyanion hole) stands out as distinctive amongst the carbapenemases. This length of this pocket is determined by the position of the OE2 atom of E166 and N atom of residue 237 which are located on opposite ends of this pocket each making one hydrogen bond with one of the water molecules. This space reduction in pocket length is quantified by the observed 0.5-1.9Å shortening of the
N:T237-OE2:E166 distance (Table 5.2). Residue E166 in itself is shifted by 0.3-0.9Å in the carbapenemases compared to non-carbapenemases (Table 5.3). This pocket is flanked by S70 whose relative repositioning of 0.5-0.8Å in carbapenemases (Table 5.3) further decreases the size of the pocket for the two waters as evidenced by the 0.5-1.4Å reduction of the CA:S70-CA:237 distance in carbapenemases relative to the non- carbapenemases (Table 5.2). S70 is therefore positioned less deep in the active site in carbapenemases. Secondly, there is increased space adjacent to the water pocket by concurrent shifts of N132 and N170 in roughly opposite directions. This is measured by the 0.6-0.9Å increase in distance between the CA atoms of N170 and N132 for carbapenemases (Table 5.2). Thirdly, a reoriented carbonyl group of C238 due to C69-
- 97 - C238 disulfide bond and 1 residue insertion after C238 is observed. Lastly, the 0.4-0.8Å relative shift of the OG atom of S130 residue, the 0.6-1.7Å relative shift of the OG1 atom of Thr235, and the 0.8-1.5Å relative shift of residue T216 are also unique features amongst the carbapenemases (Table 5.3).
The change listed in our first observation result in decreased space for the usually present waters Wat1 and Wat2 and has a remarkable effect on the number of waters present in the active sites of the carbapenemases depending on the position of S70. In
KPC-2, the side chain of S70 partially blocks the oxyanion hole and this structure therefore lacks Wat2 (Figure 5.5B). In SME-1, S70 points into the direction of the deacylation water cavity and this structure therefore lacks Wat1 yet does harbor the oxyanion hole Wat2. Only NMC-A has both Wat1 and Wat2 present in the active site since S70 has shifted somewhat in NMC-A less compared to the other two carbapenemases (Figure 5.5B). In non-carbapenemases such as SHV-1 and TEM-1, these waters are always present in their apo structures (Figure 5.5B). Since both cavities need to be available during catalysis (Wat2 cavity for the carbonyl oxygen atom of carbapenem during acylation and Wat1 for subsequent deacylation), we speculate that
S70 will likely adopt different conformations during catalysis to free these cavities in a successive manner. Increasing S70 movement is likely made possible by the more protruding S70 position in the active sites of carbapenemases (Figure 5.5B).
The above listed changes have potential consequences for the ability of carbapenemases to hydrolyze imipenem and cefoxitin. First, we hypothesize that having
- 98 - S70 positioned in a more shallow position means that the β-lactam ring of carbapenems does not have to enter the catalytic cleft as deep which would normally be sterically hampered by the 6α-substituent of carbapenems or 7α-substituent of cephamycins. To illustrate this, we have used the structure of the cephalothin:AmpC Michaelis-Menten substrate complex complex and superimposed it onto KPC-2 (Figures 4 and 6) (Beadle et al., 2002). Although AmpC is a Class C β-lactamase, it is by our knowledge the only known substrate:serine-β-lactamase complex and many key active site residues are conserved between Class C and Class A β-lactamases (i.e. S70, K73, N132, K234, G236,
T235, and A/T237). This substrate complex superposition provides a good starting point for understanding the significance of the observed shifts with respect to carbapenems and cephamycin hydrolysis, in particular since cephalothin is structurally related to the cephamycin cefoxitin as it only lacks the 7α-O-CH3 moiety (Figure 5.1).
The superposition revealed that the carboxyl moiety of cephalothin is in a similar position as the carboxyl moiety of bicine, an observation that also strengthens the relevance of bicine’s active site interactions. The observed shift of S70 being positioned less deep in the active site would allow the cefoxitin to approach the active site at a slightly different angle such that its 7α-O-CH3 moiety shifts to a wider area of the active site to minimize steric clashes (Figure 5.6A). In addition to this approximate in-plane reorientation of cefoxitin, cefoxitin is postulated to undergo an additional movement perpendicular to the first rotation (Figure 5.6B). This second rotation is likely due to the concerted shifts of S130 and T235 (Figure 5.5B) which are postulated to provide key anchoring hydrogen bonds with the carboxyl group of cefoxitin as extrapolated from the
- 99 - initial superposition of the AmpC complex with KPC-2 (Figure 5.4), the bicine interactions with KPC-2, and that the carboxyl moiety of cefoxitin forms a hydrogen bond with T316 in AmpC which is the structural equivalent of T235 in KPC-2 (Beadle et al., 2002). In addition, the carboxyl moiety of the inhibitor 6α-(hydroxypropyl)- penicillanate when bound to NMC-A forms similar interactions with T235, S237, S130, and K73 although this a covalently acyl-enzyme complex and not a Michaelis-Menten complex (Mourey et al., 1998). Both S130 and T235 are shifted such that the carboxyl group and the rest of the ligand might undergo a counter-clock wise rotation as indicated in Figure 5.6B. The active site is wide enough to accommodate such a reorientation of cefoxitin which likely results in providing additional space for the 7α-OCH3 group. An interesting observation is that the carboxyl moiety of bicine, and presumably also cefoxitin, also interacts with T237, in addition to S130 and T235 (Figures 4 and 6B). The importance for the Oγ atom at position 237 has been noted since only a S or T at this position could maintain carbapenemase activity (Sougakoff et al., 1999;Majiduddin,
Palzkill, 2005). Loss of this hydroxyl in SME-1 leads to a 5-fold decrease in kcat for imipenem hydrolysis (Sougakoff et al., 1999). This suggests that the Oγ of residue 237 could indeed be critical for orienting cefoxitin, and imipenem, via interaction with their carboxyl moieties thereby improving the catalytic efficiency of their hydrolysis.
Residues in the immediate vicinity of T237 are R220 and H274 with which it forms a 2.7 Å hydrogen bond and 3.4 Å van der Waals interaction, respectively. As noted above, all three residues are changed in SHV-1 and TEM-1 further emphasizing the importance of this region for carbapenemase activity. R220 residue provides, in addition
- 100 - to its hydrogen bond with T237, also a ~4 Å electrostatic interaction and a water- mediated interaction with the carboxyl moiety of bicine. In KPC-3, residue H274 is mutated to a tyrosine which leads to a ~6-fold enhancement in kcat/Km for imipenem compared to KPC-2 (and KPC-1) (Alba et al., 2005). Both R220 and H274 are therefore postulated to have an effect on substrate binding either electrostatically, or indirectly via correctly positioning either T237 and/or the Wat3 water molecule (see Figure 5.4). In summary, the ability of carbapenemases to accommodate α-substituents present in carbapenems and cephamycins is postulated to result from multiple active site adjustments such that these substrates can access the active site in a somewhat different orientation thereby repositioning their α-substituents to a wider part of the active site.
Shifts of nearby N170 and N132 in particular likely provide additional room as well. The active site shifts are supported by conserved residue changes in carbapenemases in particular near the postulated carboxyl binding site to provide additional direct or indirect interaction strength.
The perimeter of the active site of carbapenemases contains an additional residue that merit consideration. This residue is W105 which in the SME-1 carbapenemase and other class A β-lactamases has been found to have a strong preference for aromatic residues (Majiduddin, Palzkill, 2005). This aromatic residue at position 105 could provide favorable stacking interactions with the carbapenem substrate in the Michaelis-
Menten complex (Bethel et al., 2006;Doucet et al., 2004) as is also evident from the cefoxitin superposition (Figures 4 and 6). SME-1 and NMC-A contains a histidine at this position whereas KPC-2 contains a tryptophan but even non-carbapenemases usually
- 101 - have an aromatic residue at this position (Figure 5.5B). It is a possibility that either a H or
W at this position could provide an hydrogen bond with the substrate in addition to the postulated stacking interaction. Residues T217 and P104 (F in NMC-A and Y in SME-1) are located a bit more distant compared to W105 yet could potentially play a role in interacting with the different substituents of carbapenems and cephamycins.
In addition to the postulated changes in carbapenemases to reorient the carbapenem and cephamycin substrates such that their α-substituents can be accommodated in a productive Michaelis-Menten complex, carbapenemases have also evolved to not get inhibited by long-lived reaction intermediates of these substrates. For example, TEM-1 has been shown to react with imipenem albeit with low affinity.
However, imipenem’s acyl-enzyme intermediate becomes trapped in the active site of
TEM-1 due to displacement of the deacylation water and the repositioning of the β- lactam carbonyl outside the oxyanion hole, interacting with S130 instead (Maveyraud et al., 1998). This non-productive deacylation configuration is likely due to imipenem’s 6α- hydroxy-ethyl moiety which makes a hydrogen bond with N132. In the carbapenemases, such a trapped covalently-bound imipenem configuration is likely avoided by the simultaneous shift of N132 and S70 in somewhat opposite directions, limiting such
N132-mediated intermediate-stabilizing interactions and favoring efficient carbapenem hydrolysis (Figure 5.5B). Thus, carbapenemases are not only able to accommodate the bulkier carbapenems in the initial binding step but also avoid being inhibited by undesired long-lived intermediates. This could be aided by the postulated increased flexibility of S70. The S70 flexibility could even have a role in allowing the carbapenems
- 102 - and cephamycins to readjust to accommodate the α-substituent group during the initial
Michaelis-Menten complex.
In summary, we present the structure of KPC-2, a clinically important carbapenemase present in K. pneumoniae and other nosocomial pathogens. This analysis provides insight into the unique properties of this β-lactamase and explains its ability to hydrolyze imipenem and other carbapenems, and cephamycin, antibiotics that constitute our “last line” of defense against β-lactam resistant bacteria. Our findings also demonstrate that apparently subtle changes in active site topology (0.5 to 0.8 Å relocation of the catalytic S70) and other shifts in conserved amino acid positions in class A β- lactamases have a profound effect on substrate specificity likely by allowing the substrates to bind in a slightly different angle to alleviate steric hindrance. The process of new β-lactam drug discovery must anticipate these unexpected consequences of protein remodeling. It is important to recall that the carboxyl moiety of bicine in KPC-2 is in an almost identical position as the carboxyl moiety of our previously designed SA2-13 inhibitor when bound to SHV-1 (Padayatti et al., 2006). This suggests that the inhibition strategy of trans-enamine stabilization using carboxyl-linker penam sulfone derivatives could potentially be applicable to carbapenemases.
- 103 -
Figure 5.1. Schematic diagram of penicillin G, a carbapenem (imipenem), cephamycin
(cefoxitin), a cephalosporin (cephalothin), and bicine. The bond that is broken during the acylation step involving S70’s nucleophilic attack (curved grey dotted arrow) is shown only for penicillin G (straight dotted grey line between C7 and N4).
- 104 -
Figure 5.2. (A) Electron density of a region in the vicinity of the active site of KPC-2.
1.85 Å resolution |Fo|-|Fc| simulated annealing omit map contoured at 2.5 σ is depicted.
(B) Electron density for bicine in the active site of KPC-2. |Fo|-|Fc| simulated annealing omit electron density is contoured at 2.5 σ.
- 105 -
Figure 5.3. (A) Structure of KPC-2 β-lactamase. The secondary structure elements are depicted as yellow (β-strands), red (α-helices), and coil (green). To indicate the position of the active site, the positions of the catalytic residues S70 (magenta), S130 (orange), and E166 (green) are labeled. Bicine is color coded by atom type with the carbon atoms shown in blue. (B) Superposition of three KPC-2 molecules present in the asymmetric unit. The three KPC-2 molecules are depicted in a Cα trace. Active site residues and bicine molecules are shown in stick representation and labeled accordingly.
- 106 -
Figure 5.4. (A) Stereo figure depicting the active site of KPC-2 with bicine and a superpositioned cephalosporin. Bicine (thick dark sticks) and the cephalosporin cephalothin (thin dark sticks) are depicted. The position of cephalothin was obtained by superimposing the AmpC:cephalothin substrate complex (PDBid 1KVL) (Beadle et al.,
2002) with KPC-2. The Cα atoms used in the superpositioning are KPC-2 residues 69-73,
132, and 235-238 onto AmpC residues 63-67, 152, and 316-319 (superposition resulted in an r.m.s.d. of 0.67Å for the 10 Cα atoms). Hydrogen bonds with bicine are shown as dashed lines. The carbon position of cephalothin in which cefoxitin has a 7α-OCH3 substituent is labeled with a ‘7’. (B) Schematic diagram of interactions of bicine in the active site of KPC-2. Hydrogen bonds are depicted by dashed lines; van der Waals interactions with W105 are shown by a curved line with perpendicular lines. Hydrogen bond distances are listed in Å.
- 107 -
Figure 5.5. (A) Superposition of class A carbapenemases. The position of the active site is indicated by depicting residues S70 and Y105 as well as the flanking Ω-loop and bicine
(in stick representation with cold colored carbon atoms). A couple of loops are deviating signicantly between KPC-2 and NMC-A and SME-1 and are labeled (near residues 88 and 98). (B) Superposition of the active sites of the carbapenemases and non- carbapenemase β-lactamases. Superpositioned are KPC-2 (dark blue), NMC-A (light blue), and SME-1 (magenta) and non-carbapenemases SHV-1 (red) and TEM-1 (orange).
Green arrows highlight common shifts comparing the carbapenemase and non- carbapenemase structures. Two key clusters of waters are circled: the deacylation water primed by E166 and N170 (Wat1) and the water occupying the oxyanion hole formed by backbone nitrogens of residues 70 and 237 (Wat2). The following 11 Cα atoms were used to superposition the active sites of the β-lactamases: 69, 70, 73, 105, 130, 132, 166,
170, 234, 236, and 237.
- 108 -
Figure 5.6. Active site adjustments of KPC-2 and their postulated role in accommodating cefoxitin. The initial position of cefoxitin is obtained after superpositioning of the KPC-2 and AmpC:cephalothin active sites and adding the O-CH3 moiety to cephalothin’s 7α position to form cefoxitin (yellow). Cefoxitin was reoriented (green), guided by observed active site readjustments, to alleviate steric clashes with its 7α-O-CH3 moiety. In addition, the conformation of its 3-substituent was adjusted as well to alleviate steric clashes. A, view from the bottom of the active site. Observed key shifts in KPC-2 relative to non-carbapenemases are indicated by green arrows. The shift of residue N132 (faint in background) likely provides additional space for the 7α-O-CH3 substituent of cefoxitin.
The observed shift of S70 to a more outward position allows a less deep active site penetration of the β-lactam ring of cefoxitin. This could result in a ligand reorientation
(curved arrow) such that the 7α-O-CH3 moiety shifts (red arrow) thereby further alleviating steric clashes. B, side view obtained by an approximate 90º rotation along the horizontal axis compared to the view in A. This view shows the postulated effects of the concerted shifts of T235 and S130 (green arrows) in possibly reorienting the carboxyl
- 109 - moiety (in direction of the small curved arrows), and the rest of cefoxitin (in the direction of the larger curved arrow), in a counter-clock wise fashion to reposition its 7α-O-CH3 group (red arrow) to a wider region of the active site to prevent steric clashes. The presence of the Ser/Thr at position 237 in carbapenemases could provide an additional stabilizing hydrogen bond for the carboxyl moiety of cefoxitin to aid in this postulated reorientation (SHV-1 and TEM-1 have an Ala at this position). The described shifts and reorientation for cefoxitin are also postulated to occur for carbapenems.
- 110 - Table 5.1. Data collection and refinement statistics for KPC-2 structure
Data Collection Space group P31 Unit cell dimensions (Å) 116.25 116.25 52.00 90 90 120 Wavelength (Å) 0.97 Resolution (Å) 30-1.85 (1.92-1.85) Redundancy 3.2 Data cut-off (σ) -3.0 (default) Unique reflections 64,107 /<σ(I)> 19.7 (2.3) Rmerge (%) 6.4 (34.4) Completeness (%) 95.6 (90.8) Refinement Resolution range (Å) 30-1.85 (1.93-1.85) Atoms in asymmetric unit 6,174 R-factor (%) 14.9 (24.9) R-free (%) 19.0 (26.0) RMSD deviations from ideality Bond lengths (Å) 0.0065 Angles (º) 1.29 Average temperature factors (Å2) Protein 25.6 Bicine 35.3 Waters 26.6 Ramanchandran plot statistics Residues in -most favored regions 90.2% -additional allowed regions 9.2% -in generously allowed regions 0.6% -disallowed regions 0%
- 111 - Table 5.2 Active site distance changes of carbapenemases and non-carbapenemases.
Distances (Å)
Resolution ESD coordinate error N:T237- CD:N170- CA:S70-
(Å) using Luzzati (Å) OE2:E166 CD:N132 CA:237
Carbapenemases
KPC-2 1.85 0.17 7.1 7.3 5.8
NMC-A 1.64 0.18 8.1 7.5 6.2
SME-1 2.1 NDa 7.8 7.3 6.3
Non-carbapenemases
SHV-1 1.98 0.2 9.0 6.7 7.2
TEM-1 1.9 0.17 8.6 6.6 6.8
aCoordinate error is not listed for SME-1 but is likely similar to the 0.2Å value for SHV-1 since their resolution and refined R/Rfree values are similar.
- 112 - Table 5.3. Relative shifts in active site residues of carbapenemases compared to non- carbapenemases
Shifts (in Å) relative to SHV-1 (and TEM-1)
OG:S130 OG1:T235 CA:S70 CA:N170 CA:E166 CA:N132 CA:T/V216
KPC-2 0.6(0.5)1 1.7(1.0) 0.8(0.9) 0.6(0.5) 0.9(0.9) 0.5(0.5) 1.0(0.8)
NMC-A 0.7(0.4) 1.2(0.6) 0.5(0.5) 0.5(0.4) 0.4(0.3) 0.8(0.7) 1.5(1.3)
SME-1 0.8(0.6) 1.2(0.6) 0.7(0.7) 0.3(0.2) 0.6(0.6) 0.8(0.7) 1.5(1.3)
1Carbapenemase shifts relative to the TEM-1 structure are all listed in parenthesis
- 113 - CHAPTER 6
Crystal Structures of KPC-2 β-Lactamase in Complex
with 3-NPBA and PSR3-226
6.1 Introduction
KPC-2 β-lactamase is a potent carbapenemase and bacteria expressing this enzyme cause a strong clinical threat against carbapenem antibiotics. Furthermore, KPC-
2 has been proven to be a difficult target to develop inhibitors against (more details in
CHAPTER 1) thus furthering the need to structurally investigate different modes of inhibition of this enzyme. In this chapter we describe two new KPC-2 inhibitor complex structures.
Boron-containing compounds, either isolated from natural sources or chemically synthesized, have been studied for their biological and physiological functions
(Dembitsky et al., 2011). Due to their unique electronic structures, boronic acid compounds have enormous potential to be developed as pharmaceutical agents (Yang et al., 2003). Boronic acids have proven to be effective β-lactamase inhibitors functioning as reversible transition state analogues (Dembitsky et al., 2011). Series of boronic acids compounds as β-lactamase inhibitors have been studied, including aromatic boronic acids, acylglycyl boronic acids bearing side chains characteristic of penicillins and cephalosporins (R1 group) and sulfonamide boronic acids (Eidam et al., 2010). These boronic acid compounds, being non-β-lactam inhibitors, have the potential advantages of evading common β-lactam based resistance mechanism: loss of porin or porin mutation, recruitment of active efflux pumps, and inactivation by β-lactamases (Thomson,
- 114 - Bonomo, 2005). Among these the most common resistance mechanism is the expression of β-lactamases. In addition, β-lactam-based antibiotics and inhibitors could up-regulate
AmpC β-lactamase production (Bennett, Chopra, 1993). A number of small aromatic boronic acid compounds have been structurally characterized with Class C AmpC β- lactamase to map active site interactions as well as develop more potent inhibitors; one compound yielded a 27nM affinity for E. coli AmpC (Powers, Shoichet, 2002;Powers et al., 1999;Usher et al., 1998;Weston et al., 1998). In this chapter we crystallized 3- nitrophenyl boronic acid complexed with KPC-2 and tested its antimicrobial activity.
As a penam sulfone, PSR3-226 is a derivative of SA2-13 with a difference in the
C2 substitution moiety (Figure 6.1). SA2-13 is a rationally designed β-lactamase inhibitor which is a good inhibitor for SHV-1 β-lactamase (Padayatti et al., 2006). PSR3-226 was a modification to probe different moieties of SA2-13-like molecules to hopefully improve affinity and cell entry. In this chapter, we characterized PSR3-226 in complex with KPC-
2. Our structure reveals implications for further inhibitor improvement.
6.2 Materials and Methods
6.2.1 Subcloning
In order to purify KPC-2 at a large scale for crystallization and soaking experiments, we made a C-terminal truncated version of KPC-2 as previously described
(Petrella et al., 2008) although in a slightly different manner. The pBR322-catI-blaKPC-
2 vector in Escherichia coli DH10B cells is a kind gift of Dr. Fred Tenover, the Centers of Disease Control and Prevention, Atlanta GA. Two primers were ordered containing
- 115 - the restriction sites NdeI and EcoRI. The sequence of the forward primer KPC- NdeI is
5’-GAATTCCATATGTCACTGTATCGCCGTCTAGTT-3’. The sequence of the reverse primer KPC- EcoRI is 5’-CCGGAATTCTTAGCCCAATCCCTCGAG -3’. The sequence encoding the last four residues V292-N293-G294-Q295 was deleted. The gene encoding the C-termeinal truncated KPC-2 was PCR amplified using pfx polymerase, gel purified using QIAquick Gel Extraction kit, and ligated into the Nde-I and EcoRI- restricted sites of the pET30a vector with an insert-vector ratio of 5-to-1. The plasmid was introduced to DH5α and sequenced confirmed that it contained the gene of KPC-2 with the last residue being G291. Then the plasmid was transformed into E. coli BL21
(DE3) competent cells for large scale expression.
6.2.2 Expression and purification
Six liters of LB broth (containing 30µg/mL kanamycin) were inoculated with 3% overnight culture and were grown at 37OC until the OD600 reaches 0.5-0.6.
Subsequently, the temperature was switched to 20OC and the expression was inducted with 0.4mM IPTG and continued to grow overnight. Cells were pelleted by centrifugation and were subsequently stored at -80OC until use. Cell pellets were thawed and resuspended in 10mM Tris buffer pH 7.0 followed by lysis by sonication. KPC-2 was purified by phenylboronate affinity column and gel filtratraion as previously described
(Ke et al., 2007). Briefly, the phenylboronate affinity column was pre-equilibrated with
10mM Tris buffer pH 7.0 and then was passed through the sterilized filtered supernatant.
The column was washed with 10mM Tris buffer pH 7.0/0.5M NaCl and then KPC-2 was eluted with 0.5M Boronate pH 7.0/0.5M NaCl. KPC-2 was further purified by passing the
- 116 - protein through a Superdex 75 column (GE Biosciences) in a buffer containing 40mM
Bis Tris pH5.9. SDS-PAGE analysis indicated that the homogeneity of KPC-2 was over
95%. The protein was subsequently concentrated to 22mg/ml in 40mM Bis Tris pH5.9.
The concentration was measured via UV280 absorbance with the calculated ε280 of 1.3863
(mg/ml)/cm. About 60mg pure KPC-2 protein was obtained from 6L culture.
6.2.3 Crystallization and soaking
Sparse matrix crystallization screening and the previously published KPC-2 crystallization conditions (Petrella et al., 2008;Ke et al., 2007) were investigated. The best crystals were grown from 20% PEG6K in 100mM KSCN and 100mM citrate (pH 4) at 20OC using vapor diffusion sitting drop technique and the protein to reservoir volume ratio was 1:1. To obtain an inhibitor complexed KPC-2 structure, we initially tried to avoid citrate buffer in the soaking and freezing solutions since citrate might compete with the inhibitor in the active site (Petrella et al., 2008). The crystals were soaked in a solution containing 50 mM 3-NPBA in 25% PEG 6K and 100 mM Tris-Cl pH 7.2 overnight and were used to collect a 1.62Å data set of the 3-NPBA bound structure. To obtain the PSR3-226 bound structure, we needed to keep the citrate buffer as otherwise the crystal would dissolve. Therefore, a KPC-2 crystal was soaked in a solution containing 50 mM PSR3-226 with 25% PEG6K and 100 mM citrate pH 4.0. The longest soaking time we managed to achieve was 2 hours and 45 minutes. At longer soaking times the crystals disintegrated. The soaked crystals were cryo-protected with 20% ethylene glycol in the corresponding inhibitor and soaking solution and flash frozen in liquid nitrogen prior to data collection.
- 117 - 6.2.4 Data collection and structure determination
Data of the KPC-2: 3-NPBA complex were collected at the Stanford Synchrotron
Radiation Light Source (SSRL) BL11-1. Data of the KPC-2: PSR3-226 complex were collected at the Advanced Photon Source (APS) 23-ID. Both data sets were processed using HKL2000 (Otwinowski, Minor, 1997). The KPC-2: 3-NPBA structure was determined by molecular replacement with the program Phaser (McCoy et al., 2007c) using chain A of the C-ter truncated KPC-2 structure (Petrella et al., 2008) (PDB 3C5A) as the search model. Crystallographic refinement was performed using REFMAC
(Murshudov et al., 1997) and model building was done using COOT (Emsley, Cowtan,
2004). The KPC-2: PSR3-226 structure was determined by starting with the isomorphous crystal structure of C-terminally truncated KPC-2 (PDB 3C5A) as well as iterative rounds of Refmac refinement and COOT model rebuilding. The PRODRG2 server
(Schuttelkopf, van Aalten, 2004c) was used to obtain the parameter and topology files for the chemical structures built in the active site including the 3-NPBA and the trans- enamine intermediate of PSR3-266. During refinement, the weighting scheme for geometry and X-ray terms and Bfactor restraint were manually adjusted. Crystallographic refinement was monitored using the program DDQ (van den Akker, Hol, 1999e) and the final model quality was assessed using PROCHECK (Laskowski et al., 2001). Data collection and refinement statistics are shown in Table 6.2.
6.3 Results and Discussions
6.3.1 KPC-2:3-NPBA structure.
- 118 - The X-ray crystal structure of KPC-2:3-NPBA was determined to 1.62Å resolution. The unbiased omit Fo-Fc map contoured at 2.5σ clearly reveals a ligand covalently attached to the Oγ atom of the catalytic Ser70 residues (Figure 6.2A). A 3-
NPBA molecule fits well into the electron density with an occupancy of 1.0 (Figure
6.2A). Active site water molecules were examined carefully and refined. The final model has a Rwork of 16.0% and an Rfree of 19.2%. All the statistics are listed in Table 6.2.
The overall 3-NPBA bound structure resembles that of the wild-type KPC-2 and the C-terminally truncated structures with a RMSD of 0.574 and 0.226Å respectively. 3-
NPBA bound in the active site was found to adopt a conformation that acts as a deacylation transition state inhibitor against KPC-2. Such a boronic acid transition state analog conformation was described previously for larger BATSI compounds when bound to SHV-1 β-lactamase (Ke et al., 2011) (Figure 6.4A). Such a conformation entails that one of the boron hydroxyl group is positioned in the oxyanion hole and the other one displacing the deacylation water. The 3-NPBA interacts with the KPC-2 active site through the following interactions: the covalent bond formation between Oγ-Ser70 and boron atom of 3-NPBA; hydrogen bond network with the boron hydroxyl groups; cation interactions of the phenyl ring of 3-NPBA with Asn132 and Lys73; the π-π interaction of the phenyl ring of 3-NPBA with Trp105; and a water mediated interaction with the nitro group of 3-NPBA (Figure 6.3A).
3-NPBA is an inhibitor for both Class A KPC-2 β-lactamase with a Ki value of 1
µM, which is better than that of Ceftazidime BATSI and Cefoperazone BASTI.
- 119 - Moreover, 3-NPBA is able to inhibit Class C Amp-C β-lactamase with a micro molar Ki value (Table 6.1). Furthermore, 3-NPBA also has microbiological activity as demonstrated by the disc diffusion assay (personal communication, Dr. Bonomo). The
KPC-2:3-NPBA structure is therefore a potential starting point for future drug design efforts to optimize this boronic acid transition state inhibitor.
6.3.2 KPC-2:PSR3-226 structure.
The X-ray crystal structure of KPC-2:PSR3-226 complex was determined to
1.26Å resolution. The unbiased omit Fo-Fc map contoured at 2.5σ revealed density for a ligand covalently attached to the Oγ atom of the catalytic Ser70 residue (Figure 6.2B). At the later stages of refinement, we carefully modeled a trans-enamine intermediate in the
KPC-2 active site as the inhibitor torsion angle defined by the atoms CAJ-CAK=CAL-N was close to 180º (it subsequently refined to 179.92°). The density surrounding the covalent bond between Oγ of Ser70 and refinement of the intermediate at different occupancies indicated that the occupancy of this intermediate is 0.7. Clear density for the atoms of this intermediate was seen up until its carboxylate group yet the electron density for rest of the tail of the compound is lacking indicating that this tail is flexible including the sulfone functional group and the R2 group. The apparent flexibility of this part of the compound was also evident in the refined B factors, which is consistent with its poor density. Citrate molecules with two conformations were also modeled nearby the PSR3-
226, of which 5 citrate atoms had strong density (Figure 6.2C). The position of citrate is also consistent with where it showed previously in the KPC-2 structure (Petrella et al.,
2008). By careful examination and adjustment at the very end of the refinement, we
- 120 - placed a water molecule with occupancy of 0.3 in the oxyanion hole and a deacylation water molecule with occupancy of 0.5 to account for the extra density at these corresponding positions. The inclusion of the PSR3-226 trans-enamine intermediate brought down the R/Rfree considerably indicating correct placement. Throughout the refinement, the occupancy of the modeled PSR3-226 intermediate was kept at 0.7. The final model has a Rwork of 15.4% and an Rfree of 16.9% and refinenment statistics are listed in Table 6.2.
The overall structure resembles the wide-type KPC-2 and the C-terminally truncated structures with a RMSD of 0.591 and 0.158Å respectively. The active site has also undergone little change except for residues W105 and S130 which now have alternative conformations. The disulfide bond between residues 68 and 237 is present but likely not 100% formed as there is some weak difference density for alternate positions of the sulfur atoms. This minor presence of a reduced disulfide could perhaps be due to X- ray radiation damage. The PSR3-226 forms a linear trans-enamine intermediate in the active site with the carbonyl being positioned in the oxyanion hole as was seen before for
SA2-13 and tazobactam (Padayatti et al., 2006) (Figure 6.4B). The C3 carboxylate group is in hydrogen-bond distance with N132, E166 and N170. There is an intra-molecular hydrogen bond. And PSR3-226’s sulfone group is positioned near residues P104 and
N132 whereas the C2 substituent points toward the bulk solvent and makes no contact with the enzyme. From Figure 6.3B, we could also see that in the presence of the PSR3-
226 intermediate, W105 was likely forced to adopt an alternative conformation to avoid steric clashes with the inhibitor.
- 121 -
PSR3-226 has improved affinity for both SHV-1 and KPC-2 compared to that of tazobactam and SA2-13 (Table 6.1), which suggests that PSR3-226 likely more readily forms a pre-acylation Michaelis-Menten complex. When inhibiting SHV-1, PSR3-226 has comparable first order rate constant to tazobactam. Regarding KPC-2, the first order rate constant of PSR3-226 for acylation is 3 fold faster than that for SHV-1. These results indicate that PSR3-226 is not only a more potent inhibitor compared with SA2-13 but that PSR3-226 is especially promising as a lead KPC-2 inhibitor due to it s strong microbiological effect in bacteria expressing KPC-2 (personal communication with Dr.
Bonomo).
6.4 Conclusion
In this chapter, we have determined the complex structures of two potent inhibitors (3-NPBA and PSR3-226) bound to KPC-2. The nature of these two compounds and their modes of inhibition are very dissimilar. While 3-NPBA is a boronic acid transition state inhibitor, PSR3-226 is a penem compound that forms a trans-enamine intermediate. These two KPC-2 complex structures and the observed active site interactions can be used for future inhibitor design studies against the clinically important
KPC-2 β-lactamase.
- 122 -
Figure 6.1. Chemical structures of the inhibitors. 3-NPBA refers to 3-nitrophenlboronic acid. The reaction scheme is taken from Padayatti et al (Padayatti et al., 2006).
- 123 - A
B
C
- 124 -
Figure 6.2. Unbiased omit Fo-Fc maps contoured at 2.5σ are depicted (A) 3NPBA in the active site of KPC-2 β-lactamase; (B) trans-enamine intermediate of PSR-3-226 in the active site of KPC-2 β-lactamase. Citrate molecules in Figure B are omitted for clarity;
(C) Citrate molecules. KPC-2 β-lactamase carbon atoms are colored gray80. Inhibitor carbon atoms are colored black. Nitrogen atoms gray20; oxygen atoms gray40, sulfur atom gray60 and boron atoms gray70. Non-carbon atoms of the inhibitor are depicted in sphere with the size from big to small as: sulfur, oxygen, nitrogen and boron atoms. wat231, wat313 and wat316 are active site water molecules and depicted as spheres.
- 125 - A
B
Figure 6.3. Stereo view of interactions of the bound inhibitors within KPC-2 β-lactamase active site. (A) 3-NPBA; (B) PSR-3-226; Citrate molecules in Figure B are omitted for simplicity. Dashed black lines indicated hydrogen bonds.
- 126 - A
B
- 127 - Figure 6.4. (A) Active site superposition of KPC2:3-NPBA structure with SHV-
1:cefoperazone BATSI structure (PDB ID: 3MKF). KPC2:PSR3-226 is colored black while SHV-1:SA2-13 is colored white. (B) Active site superposition of KPC2:PSR3-226 structure with SHV-1:SA2-13 structure (PDB ID: 2H5S). Residues used for superposition include 70, 73,105,130,132,166,170 and 234-237. KPC2:PSR3-226 is colored black while SHV-1:cefoperazone BATSI and SHV-1:SA2-13 are colored white. Enzymes are shown as transparent cartoon representation. Inhibitor carbon atoms are colored correspondingly and shown as lines. Nitrogen atoms gray20; oxygen atoms gray40, sulfur atom gray60 and boron atoms gray70. Water molecules are omitted for simplicity.
- 128 - Table 6.1. Inhibition and kinetics data
KPC-2: ceftazidime BATSI Ki = 54 ± 8 µM *
KPC-2: cefoperazone BATSI Ki = 2.0 ± 0.3 µM *
KPC-2: 3-NPBA Ki = 1 µM *
AmpC: 3-NPBA Ki = 1.7 µM (Weston et al., 1998)
-1 SHV-1: PSR3-226 Kd= 4.43 ± 0.22 µM kinact = 0.10 ± 0.01(s ) *
-1 KPC-2: PSR3-226 Kd= 3.8 ± 0.4 µM kinact = 0.034 ± 0.003 (s ) *
-1 SHV-1: tazobactam Kd= 0.1 ± 0.025 µM kinact = 0.10 ± 0.006(s )
(Padayatti et al., 2006)
-1 SHV-1: SA2-13 Kd= 1.7 ± 0.1 µM kinact = 0.05 ± 0.001(s )
(Padayatti et al., 2006)
(*, preliminary data from Dr. Bonomo’s lab)
- 129 - Table 6.2. Data collection and refinement statistics
3NPBA : KPC-2 PSR-3-226 : KPC-2 Data collection
space group p212121 p212121 cell dimensions a, b, c (Å) 50.348 66.558 73.598 49.051 66.288 71.776 α, β, γ (deg) 90.000 90.000 90.000 90.000 90.000 90.000
wavelength (Å) 0.97945 0.98400 resolution (Å) 50.00-1.62 (1.68-1.62) 50.00-1.26 (1.31-1.26) Rsym 5.9 (36.1) 7.3 (27.2) I/σI 21.3 (3.3) 23.1(3.4) Completeness (%) 91.9 (75.7) 97.9 (82.5) Redundancy 4.4 (4.4) 6.7 (2.7) Refinement Resolution range (Å) 27.77-1.62 (1.661-1.619) 33.15-1.26 (1.293-1.260) no. of reflections 28074 59322
Rwork/Rfree 16.0/19.2 (25.8/29.2) 15.4/16.9 (22.5/24.6) no. of atoms: protein, inhibitor, water, others 2010, 12, 246, 0 2013, 21, 316, 26 b rmsd bond length (Å) 0.010 0.010 bond angles (deg) 1.226 1.369 average B-factors (Å2) protein, inhibitor, water, others 15.1, 22.5, 30.6, 0 13.4, 53.0, 28.3, 73.8 Ramanchandran plot statistics (%) core regions 94.7 94.7 additional allowed regions 5.3 5.3 generously allowed regions 0.0 0.0
disallowed regions 0.0 0.0 a Numbers in parentheses refer to the highest resolution shell. b rmsd, root-mean-square deviation
- 130 - CHAPTER 7
Heterotropic Allosteric Regulation Mechanism of an
Engineered β-Lactamase-MBP Fusion Protein
7.1 Introduction
Allosteric regulation is a common mechanism cells utilize to regulate protein activity. Heterotropic allosteric proteins have a ligand binding site distant from the substrate binding site to allow conformational changes to be transmitted from the ligand regulatory binding site. Mechanisms of allosteric regulation have been studied extensively in a multi-disciplinarily approach in the recent years, including X-ray crystallographic and NMR-based structure elucidation, molecular dynamics simulations, novel relaxation dispersion NMR, nano/picosecond time-resolved X-ray crystallography
(Swain, Gierasch, 2006;Ostermeier, 2005). In addition, engineered proteins with allosteric properties have attractive applications including molecular biosensors, therapeutic proteins, and gene regulation(Swain, Gierasch, 2006;Guntas et al., 2005).
Mimicking non-homologous recombination via domain/module combinations and rearrangements is a relatively novel approach to develop complex new functions regarding molecular recognition and regulation (Koide, 2009). Such an insertional protein engineering approach can lead to development of analytical molecular sensors (Ferraz et al., 2006). A successful example of this approach is the allosteric protein RG13 which is a molecular switch created by recombining the nonhomologous genes of Escherichia coli
Maltose Binding Protein (MBP) and TEM-1 β-lactamase and selected for under
- 131 - evolutionary pressure (Guntas et al., 2004). In brief, a circular permutated TEM-1 gene was randomly inserted into the gene encoding MBP. The constructs were subjected to in vitro selection against ampicillin and maltose. The resulting RG13 construct was demonstrated, in the presence of maltose, to have β-lactamase activity comparable to the wild-type TEM-1 β-lactamase but RG13’s activity is decreased 25-fold in the absence of maltose(Guntas et al., 2004). As a consequence, RG13 has also achieved its goal of in vivo maltose-dependent ampicillin resistance(Guntas et al., 2004). Intriguingly, RG13 activity was serendipitously found to be negatively regulated by zinc in a non- competitive and reversible mode, a characteristic that neither parent enzyme MBP nor
TEM-1 possesses(Liang et al., 2007).
To our knowledge, no structural knowledge of how allostery is achieved by switches obtained by non-homologous domain insertion, such as RG13, is currently present and that is the focus of this study. Initial insights into the allosteric mechanism of
RG13 were obtained by the Ostermeier’s lab with mutational studies indicating that the conformational changes regarding the domain closure angle in the MBP domain, which is maximally achieved by maltose binding, are responsible for affecting TEM-1 activity in
RG13(Guntas et al., 2004). Furthermore, the extent of this MBP subdomain hinge angle is key for RG13 functioning since mutations that are known to affect the hinge angle to a more closed conformation, reduce the maltose switching of RG13 activity (Kim,
Ostermeier, 2006). A recent NMR study compared the NMR data of RG13 in the absence/presence of maltose with data from previous NMR studies of MBP and TEM-1 and provided evidence that: (1) the individual MBP and TEM-1 domain structures in
RG13 are substantially conserved; (2) the TEM-1 active site of RG13 is relatively
- 132 - unperturbed in the presence of maltose; (3) and in the absence of maltose the active site of RG13 is slightly perturbed with possible displacement of several residues(Wright et al., 2010).
We are interested in delineating the mechanism of switching of RG13 as to how the TEM-1 activity is positively regulated by the allosteric effector maltose and negatively regulated by zinc. The RG13 crystal structure presented here captures RG13 in its inhibition state with the negative regulator zinc ion bound; the structure shows that the zinc ion bridges the TEM-1 and MBP at an additional contact point away from the double cross-over linker region thereby negatively affecting the conformation of this cross-over region that is likely key for RG13 maltose switching. Furthermore, we carried out mutagenesis studies and molecular dynamics simulations to probe the RG13 structure in the absence and presence of zinc to further gain insights into zinc regulation of RG13.
7.2 Materials and Methods
7.2.1 Expression and Purification
The plasmid of RG13 expressed in malE¯ auxotroph PM9F’ Escherichia coli was a kind gift from Dr. Marc Ostermeier. RG13 was expressed and purified as previously described (Liang et al., 2007). Briefly, the expression plasmid containing RG13 was transformed into Escherichia coli strain PM9F'. Gene expression were induced by 1mM
IPTG at OD600 0.6-0.8. The culture was grown overnight at 20°C. Cells were lysed by
French press and RG13 was subsequently purified using an amylose affinity resin (New
England Biolabs) and eluted using maltose. The eluted RG13 protein was dialyzed at
4OC, to remove maltose, and subsequently concentrated to ~ 11mg/mL in 20mM Tris-Cl
- 133 - pH7.0, mixed with glycerol for storage at -80OC. Prior to crystallization, aliquots of
RG13 were thawed and subjected to gel filtration using a Superdex200 column (GE
Biosciences) that was equilibrated with 20mM Tris/HCl pH 7.2 and 500mM NaCl. RG13 was subsequently buffer exchanged to 10mM Tris/HCl pH 7.2, and concentrated to 10-
15mg/ml (as assessed by the Bradford assay).
7.2.2 Crystallization and soaking
Prior to crystallization, ZnCl2 was added to RG13 to a final concentration of 2.5 mM. Initial crystallization conditions were found by using Hampton screen kits. After optimization, single crystals were obtained by vapor diffusion sitting drop method at room temperature: a 1.0 µl drop was prepared using 0.5µl protein mixture (13.8 mg/ml
RG13 and 2.5 mM ZnCl2) and 0.5 µl reservoir solution (containing 0.2 M ammonium acetate, 0.1M Tris pH 8.5-9.5, 15-30% PEG 3350) and equilibrated over a 1 ml reservoir solution. RG13-Zn co-crystals grew to full size in one week. The cryo-protectant for crystals is reservoir solution with 33% PEG 3350 and 2.5 mM ZnCl2.
7.2.3 Data collection and structure determination
Crystals of RG13 grew in two different space groups. Diffraction data for the
RG13-Zn complex P1 and C2 spacegroup crystals were collected at the National
Synchrotron Light Source beam line X-29A and processed using HKL2000 (Minor et al.,
2000;Otwinowski, Minor, 1997). The data from the P1 space group was double checked but could not be processed in a higher symmetry space group. The structures for each of the space groups was solved by Molecular Replacement using the program Phaser
- 134 - (McCoy et al., 2007d) with two RG13 molecules in the P1 space group and one RG13 molecule in C2 space group. Based on the folding pattern of MBP and the circular permutation of RG13 (Guntas et al., 2004), four ensembles were used to carry out a
Molecular Replacement search for the two copies of RG13 molecules in the asymmetric unit of P1 space group. These four ensembles are: 1OMP subdomain-1 (PDB ID: 1OMP, residues 1-109 & 263-310), 1OMP subdomain-2 (residues 114-258 & 319-370), 1ZG4 region 1 (PDB ID: 1ZG4, residues 26-224), and 1ZG4 region 2 (PDB ID: 1ZG4, residues
245-280). The Molecular Replacement program Phaser (McCoy et al., 2007a) successfully managed to place 6 chains in the asymmetric unit of P1 space group except for the smallest fragment which was added in later manually and guided by the electron density (1ZG4 region 2). A similar molecular replacement procedure was carried out to solve the RG13 structure in the C2 space group with a single RG13 molecule in the asymmetric unit. The resulting electron density maps were manually inspected and residues were added or removed using COOT (Emsley, Cowtan, 2004). After several rounds of REFMAC (Murshudov et al., 1997) refinement and rebuilding using COOT, most residues of the full length of RG13 protein in both space groups could be built.
During the refinement, inspection of Fourier difference maps indicated the presence of very strong non-protein electron density peaks which were identified, and refined subsequently, as zinc ions. Water molecules were included in the final stages of refinement. The simulated annealing protocol in CNS (Brunger et al., 1998a) was also used in refinement. Iterative rebuilding and refinement allowed the final model to converge with an R/Rfree 23.2/29.2 for the P1 space group and 23.3/29.1 for the C2 space group (Table 7. 1 for more refinement stats). The C2 space group contains RG13 residues
- 135 - 1-316, 328-582, 585-637, 2 zinc ions, and 134 water molecules whereas the P1 space group structure contains RG13 residues 1-637, 4 zinc ions, and 490 water molecules.
PROCHECK (Laskowski et al., 2001) and DDQ (van den Akker, Hol, 1999f) were used during the model building process and to validate the final structures. The atomic coordinates and structure factors of the RG13 with zinc ions for the P1 and C2 space groups have been deposited in the Protein Data Bank under accession code 3QN4 and
3QN5, respectively.
7.2.4 Molecular Dynamics Simulation
Three Molecular Dynamics (MD) simulations were carried out using the program
Gromacs (Hess et al., 2008;Van Der Spoel et al., 2005). The wild-type simulation used the zinc-bound RG13 structure of P1 space group (monomer A including the zinc ion that is interacting with both its TEM-1 and MBP domains) as the starting structure to evaluate its solution dynamic state in the presence of zinc. To probe the structure of RG13 in the absence of zinc, a no-zinc simulation was carried out using the same RG13 monomer, with the zinc ions deleted, as the starting structure. For the wild-type-TEM-1 simulation, the crystal structure of TEM-1 β-lactamase (PDB id: 1ZG4) was used as the starting structure to serve as a control simulation. All simulations used the G43a1 force field and the complexes were immersed into Cubic box filled with spce water molecules. Counter ions (Na+) were added correspondingly to each model to neutralize any net charge. Each system was subsequently energy minimized using the steepest decent algorithm.
Following this energy minimization, 60 ps of position-restrained dynamics simulation was performed followed by molecular dynamics simulation for a time period of 17ns for
- 136 - each of the simulations. All three simulations were performed similarly as above except the RG13 wild-type simulation, which had an additional distance restraint added to maintaining the relative position of the zinc ion to its ligands (D164 & H468). The distance restraints were modeled as described previously (Manzetti et al., 2003). In brief, an energy penalty was applied whenever the Zn-ligand distance was more than 0.1nm beyond the starting model. The force was proportional to the distance beyond 0.1nm with a force constant of 1000 kJ/mol nm2. The MD simulations were carried out at the CWRU
High Performance Computing Cluster.
7.3 Results
7.3.1 Overall structure
The primary structure of RG13 is unusual in that it is a 72kD fusion protein comprised of the circularly permutated TEM-1 gene inserted into MBP at MBP residue
316 (Guntas et al., 2004). Due to the circularly permutated TEM-1 gene, new TEM-1 N- and C-termini are used to fuse this gene into the MBP gene as the original TEM-1 N- and
C- terminus are connected by a linker comprised of residues GSGGG. As a result of generating and selecting for RG13, residues 317 and 318 of MBP are no longer present and are replaced by a serine residue introduced at one of the insertion sites (Guntas et al.,
2004). In total, there are two connections between the TEM-1 and MBP domains with the schematic of the domain fusion of RG13 shown in Figure 7.1A.
- 137 - Two types of crystals of RG13 were grown in the presence of zinc. These crystals belonged to space groups P1 and C2 and were refined to 2.30 Å and 2.29 Å resolution, respectively (Table 7. 1). The RG13 structure reveals that despite the domain shuffling and different connections, RG13 contains two folded domains comprised of the regulatory MBP and the catalytic TEM-1 (Figure 7.1). The overall dimensions of each
RG13 molecule are roughly 50 × 70 × 70 Å with the TEM-1 and MBP domains lying alongside each other connected by two linkers. The two RG13 molecules in the P1 space group have a similar conformation as the root-mean-square-deviation (r.m.s.d.) for Cα atoms is 0.39Å (for 636 residues). Superpositioning of the RG13 molecule from the C2 space group onto each of two molecules in the P1 space group yields an rmsd of 1.7 Å
(619 residues) and 1.6 Å (615 residues) for molecules A and B, respectively. The electron density for most of RG13 is well resolved including for most of the TEM-1 active site
(Figure 7.2A). The two connecting loops between TEM-1 and MBP could only be resolved in the P1 space group in both monomers although these loops, not unexpectedly, had higher temperature factors than most of the rest of the protein (Figure 7.2); part of these linker regions comprising residues 317-327 and 583-584 are not included in C2 space group structure due to lack of electron density. Similarly, the newly generated linker GSGGG linker introduced to circularly permutated TEM-1 is resolved in the electron density maps (Figure 7.2D) but also has somewhat higher temperature factors compared to the cores of the two domains (Figure 7.2E).
The orientation between RG13’s TEM-1 and MBP domains in the C2 space group is about 8.5° different compared to that in the P1 space group (Figure 7.1C) indicating a
- 138 - certain flexibility in orientation between the two domains, which is not unexpected due to the presence of the somewhat more mobile linker regions and the limited contacts between the two domains. An additional contact between MBP and TEM-1 in each of the
RG13 monomers is mediated by a zinc ion.
7.3.2 Zinc binding
In addition to the two RG13 monomers in the P1 space group, there are four zinc ions present with two zinc ions bound per monomer. Similarly, there are two zinc ions present in the C2 space group structure of RG13. The zinc ions are readily identifiable being the strongest difference Fourier density features in difference density maps (Figure
7.3). The zinc ions bound to the three different RG13 monomers can be grouped in two different classes: the main zinc ion bridges both domains of RG13 interacting thus with both the TEM-1 and MBP domains whereas the second zinc only binds to H509 (H153 in
TEM-1 residue numbering shown in italics and brackets from here on forward) and H514
(H158) of the TEM-1 domain (in C2 space group). A similar zinc binding site is also present in the P1 space group involving the same H509 and H514 residues but this zinc binding site is expanded and also includes ligands from a crystallographically related
RG13 MBP domain: H39’ and Y17’ (Figure 7.3C-3D). The main zinc ion in the C2
RG13 structure bridges the TEM-1 and MBP domains via residues H468 (H112), both oxygens of residue D164 (MBP residue), residue E477 (E121)(Figure 7.3B). In the P1 structure, residue E477 has slightly shifted away and therefore the main zinc ion only interacts with H458 and both oxygens of D164 (Figure 7.3A). Despite the second zinc
- 139 - binding site likely not being functionally relevant for RG13, the observation of the additional zinc binding site indicates that RG13 has a tendency to readily bind zinc ions which will be addressed further in the Discussion section. The main zinc ion binding site in RG13 is distant from the active site being situated 20Å from the Oγ atom of the catalytic S426 residue (S70).
7.3.3 MBP domain and TEM-1 domain
MBP adopts a periplasmic binding fold and is capable of undergoing a ligand induced ‘pacman’-type subdomain closure in which the ligand maltose binds to both subdomain halves of MBP leading to a domain closure of 35° (Quiocho et al., 1997). The
MBP domain of RG13 conformation is more similar to the uncomplexed MBP structure
(PDB ID: 1OMP), as reflected in the relative low r.m.s.d. values of 1.25Å monomer A in
P1, 1.39 Å for monomer B in P1, and 1.81Å in space group C2 compared to the maltotriose complexed wild-type MBP structure (PDB ID: 3MBP) which yielded larger r.m.s.d.s. of 2.76 (P1 monA), 2.80 (P1 monB), and 2.0 Å (C2) for about 360 Cα atoms
(Figure 7.4). This is in agreement with a subdomain rotation analysis indicating that
RG13 is in a more unliganded open conformation in both P1 and C2 since it its domain closure angle is about 10° and 17°, respectively, compared to that of the uncomplexed
MBP structure (subdomain 1 of MBP is defined as 1-109 & 261-316 and subdomain 2 is defined as 110-260 & 586-637 in RG13). The above calculations and superpositioning of one of the MBP subdomains (Figure 7.4) do however indicate that the MBP domain closure angle can vary in RG13 with the C2 RG13 structure being slightly closer to that of the ligand bound MBP structure compared to the P1 RG13 monomers. Nevertheless,
- 140 - the more open conformation of the MBP domain in RG13 is consistent with it being crystallized under maltose-free conditions. The analyses also indicate that the two subdomains of MBP are still flexible in the absence of maltose and that crystallization can trap them in slightly different conformations.
The TEM-1 domain of RG13 has an overall fold similar to the wild-type TEM-1 structure (PDB ID: 1ZG4) with a core r.m.s.d. of 1.02Å (P1 molA), 1.02 Å (P1 molB), and 1.1 Å for ~240 Cα as calculated using COOT (Emsley, Cowtan, 2004). The most striking structural change in the TEM-1 domain of RG13, compared to the wt TEM-1 structure, is the relocation of the critical active site β3 strand and part of the β4 strand and connecting loop. This TEM-1 active site region in RG13 is now part of the linker connecting the MBP and TEM domains and therefore no density is observed in the region where these strands used to be in the active site of the wild-type TEM-1 structure
(Figures 5-6). The displaced section comprises TEM-1 residues 229-244 with also the preceeding residues 214-228 being in a somewhat different conformation as the TEM-1
α10 helix extends in RG13 forming a longer helix (Figure 7.6). Despite these substantial active site differences of missing active site β-strands, the positions of most other active site TEM-1 residues including S70, Y105, S130, N132, E166, and N170 (in TEM-1 residue numbering) are relatively unchanged in RG13. TEM-1 is a β-lactamase that hydrolyses bicyclic β-lactam compounds that contain a carboxyl and carbonyl moiety.
Key active site elements for TEM-1 are the catalytic serine S70, the carboxyl binding pocket, the oxy-anion hole for carbonyl oxygen, and the deacylation E166 residue. The
- 141 - β3-strand plays an important role for TEM-1 activity because it forms one of the active site walls providing part of both the carboxyl binding pocket (via S235) and oxy-anion hole for the carbonyl oxygen (via the backbone nitrogen of residue A237) which are thus no longer in the correct position in RG13. Residue K234 is also critical for β-lactamase functioning (Lenfant et al., 1991) and no longer in the native TEM-1 position (Figure 7.5-
6). Furthermore, β4-strand residue R244, which is also involved in electrostatic interactions with the β-lactam carboxyl moiety (Thomson et al., 2006), has also shifted.
This β3-β4-strand section of TEM-1 is converted into a linker between TEM-1 and MBP in the RG13 structure (Figures 1B, 5, and 6). This is due to this region’s close proximity to one of the fusion sites, i.e. residue 229, used to fuse TEM-1 and MBP (Figure
7.1)(Guntas et al., 2004). In addition to lacking some key active site features, the entry to the TEM-1 active site in RG13 is partially blocked by this displaced stretch of linker region that forms a short helix (Figures 5-6). Taken together, loss of β3-β4 strand section and steric hindrance within the active site by this displaced stretch of residues likely explains the lack of activity of our zinc bound RG13 structure.
7.3.4 Mutagenesis
The preliminary mutagenesis data was provided by Dr. Ostermeier’s lab and we included this for discussion purpose. As zinc had previously been shown to inhibit RG13 in a non-competitive, yet reversible manner with a Ki value of 2.7 µM (Guntas et al.,
2004), the crystal structure of RG13 with zinc bound points to the location of the possible inhibitory zinc binding site. To probe the importance of the crystallographically observed main zinc binding site for zinc inhibition of RG13, we mutated the zinc liganding
- 142 - residues D164 and H468 in RG13 to alanine. The resulting Ki of zinc for the RG13
D164A/H468A double mutant dropped modestly from to 8.7 ± 1.4μM for the double mutant RG13 D164A/H468A.
7.3.5 Molecular dynamics simulations of RG13
We were unfortunately not able to obtain crystals of uncomplexed zinc-free RG13 nor of that of maltose-bound RG13. To nevertheless probe the structure of RG13 in its zinc inhibited state and its non-zinc bound state, we performed MD simulations using the
Gromacs package. Three different simulations were carried out for 17ns each: a zinc bound RG13, a zinc-free RG13, and a TEM-1 control MD simulation. Backbone r.m.s.d. calculations compared to the starting structure of each reached a relative plateau indicating some (local) equilibrium had been reached (Figure 7.7). The P1 monomer A
RG13 crystal structure and the three 17ns-simulation structures were superposed onto the wild-type TEM-1 structure (PDB ID: 1ZG4) such that the relative end orientation of the
MBP domain could be compared as well as other structural changes. At 17ns, the MBP domain of zinc-RG13 MD simulation was observed to have rotated 6.5º compared with our starting RG13 crystal structure while the MBP domain of no-zinc-simulation rotated
41.2º compared with the crystal structure (Figure 7.8). The results from these simulations indicate that the main zinc binding site in RG13 plays an important role in locking the
MBP domain into a different specific orientation with respect to the TEM-1 domain. This suggests that a 3-point ‘attachment’ of the MBP and TEM-1 domains, via linker 1, linker
2, and zinc hold the two domains in a different orientation compared to having only a 2- point ‘attachment’, with two linkers only, as evidence in the zinc-free RG13 molecular
- 143 - dynamics simulation. This observation of potential inter-domain reorientation and likely concomitant repositioning of the two linkers in the absence of zinc offers the possibility that the linker regions might be relaxed enough to re-enter the TEM-1 active site to reform the critical β3-β4 strands. For comparison, we also carried out a wild-type TEM-
1-only MD simulation which indicated that only minor changes occured in the TEM-1 only MD simulation (Figure 7.8C). The MD simulations also indicated some movement in RG13 of the TEM-1 helices 1 and 12 compared to the wild-type TEM-1 structure.
These two helices are the N- and C-terminal helices of TEM-1 which are now joined via the engineered GSGGG linker as part of the circular permutation that connects the N- and
C- terminus of the TEM-1. These two helices probably shifted during the simulations as there is a gap in the active site since the β3-β4 strand section is displaced in RG13; the β4 strand was contacting this C-terminal helix in the wild-type TEM-1 structure perhaps now allowing these helices to move somewhat in the RG13 structure.
7.4 Discussion
The crystal structure of RG13 bound to the non-competitive zinc inhibitor provides insights into the heterotropic allosteric signaling of RG13. The structure reveals that the TEM-1 and MBP domains are in close proximity to each other connected via two linkers and an additional bridging zinc ion. Whereas the MBP domain is quite intact adopting a relatively open/unliganded conformation, the TEM-1 domain active site is compromised with the active site β3-β4 strand section mostly displaced from their wild- type TEM-1 positions now partially blocking substrate entry while forming a linker between the two domains. Comparison of two different space group RG13 structures
- 144 - reveals that there is still some flexibility in the orientation between the MBP and TEM-1 domains as well as within the two lobes of MBP (Figure 7.1C). Similarly, the RG13 MD simulations with zinc show somewhat comparable relative orientational variations between the TEM-1 and MBP domain (Figure 7.8). In contrast, the MD simulation of
RG13 in the absence of zinc reveals a much larger rotational freedom of these domains which reorient themselves drastically. The zinc ion therefore seems to inhibit RG13 by generating an additional anchor point and therefore acts to ‘twist-tie’ the original anchor points and linkers such that these linkers are contorted and no longer have enough slack to reinsert into the active site. In particular β3-β4 strand residues 228-244 are no longer correctly oriented to generate a productive TEM-1 active site. The β-strand expulsion and insertion is reminiscent of how the blood clotting serpin anti-thrombin is regulated (Jin et al., 1997;Schreuder et al., 1994). The zinc acts thus as a non-competitive inhibitor binding distant from the active site yet clearly affecting β-lactamase activity. We do note that although the MD RG13 simulations do reveal a large reorientation of the MBP and
TEM-1 domains and relaxing of the linker regions in the absence of zinc, the simulations are clearly not long enough to actually witness the anticipated TEM-1 active site regeneration of RG13 involving β3-β4 strand reinsertion.
7.4.1 Insights into maltose activation of RG13
From the above analyses, it is evident that RG13 is in the inhibited state due the displacement of the critical β3-β4-strand region now partially blocking the active site as this region now acts as a linker connecting the two domains. Previous kinetic studies in
- 145 - the absence of both maltose and zinc indicated that RG13 has a ~25-fold lower kcat/Km activity compared to wild-type TEM-1 with both the kcat and Km each affected about 5 fold (Liang et al., 2007;Guntas et al., 2004;Guntas et al., 2005) (Table 7. 2). Furthermore, kinetic data show that in the presence of maltose, RG13 has a restored kcat and Km to such an extent that it is comparable to the wild-type TEM-1 which is remarkable for a fusion protein, in particular in light of the RG13: zinc structure revealing a severely compromised TEM-1 active site. Moreover, a previous NMR study found that the active site of TEM-1 in RG13 is relatively unperturbed in the presence of maltose (Wright et al.,
2010). These observations suggests that RG13 has a much more TEM-1 native-like active site in the maltose-bound activated state compared to our observed zinc bound RG13 structure which likely displays the TEM-1 domain in a relative ‘off-state’. To gain insights into how an intact TEM-1 structure would fit within the RG13 framework, we modeled an intact TEM-1 molecule (with the β3-β4-strand region in the wild-type position) in close proximity to the maltose-bound MBP (Figure 7.9). Firstly, maltotriose bound (PDB id: 3MBP) and unbound (PDB id: 1OMP) MBP structures were superposed onto to the RG13-Zn crystal structure by superpositioning of residues 1-109 and 261-316 which is one of the sub-domains of MBP. Subsequently, G584 (G228) and F318 (F230) were chosen as the anchor points to attach TEM-1 to the MBP in RG13 (we left W229 in its RG13 position). In the modeled TEM-MBP RG13-like complex, the TEM-1 domain has a three dimensional location fitting reasonably well without major clashes but the
G228 anchor point is just somewhat farther away from where it is in the RG13-Zn structure (Figure 7.9B). The superpositions of the maltose-free and maltose-bound MBP indicated that maltose binding induces a shift in the helix connecting to one of the anchor
- 146 - points G584(228) therefore bringing this anchor point closer to the second anchor point
F318(F230) (Figure 7.9A). It is interesting that this shift is in the right direction and rough distance that the G584 (G228) might move to the position of where it is situated in the modeled wild-type TEM-1 structure (Figure 7.9B and 9C). Therefore, maltose binding likely tweaks this region in that the β3- β4 strand region involving TEM-1 residues 228-244 is relaxed enough to fit correctly and productively in the active site such that RG13 yields wild-type TEM-1 activity in the presence of maltose. Furthermore, we hypothesize that in maltose-free RG13, the β3-strand is likely mostly in the rough correct position as the kcat and Km only change roughly 5-fold each upon maltose binding (Table
7. 2). If the β3-strand would be missing entirely in the absence of maltose, the effect on activity would be much worse as there would be no oxyanion hole, no K234 and R244, and such an active site configuration would likely therefore also have little or no catalytic activity. For example, the mutation K234T in TEM-1 affect substrate affinity 50-fold
(Lenfant et al., 1991), and K234A in a related β-lactamase affected both Km and kcat such that the kcat/Km was decreased multiple orders of magnitude (Ellerby et al., 1990). We hypothesize that by pulling slightly on the TEM-1 β3-strand, which starts at TEM-1 residue F230, this strand is modestly shifted in maltose-free RG13 affecting both kcat
(oxyanion needed) and Km (carboxyl moiety interaction needed). In agreement with this hypothesis is that NMR studies showed that the N-terminal one third to one half of this
β3 strand is displaced in the maltose-absent NMR structure of RG13 (Wright et al.,
2010). Since this part of the β3 strand is very close to both K234 and A237 of TEM-1 located in the middle of the β3 strand (even though these two residues were assigned in the NMR spectra), subtle changes in their dynamics and position could account for the
- 147 - 25-fold maltose-dependent activity switching of RG13. By slightly pulling of this end of the N-terminal end of the β3 by a maltose-free MBP domain in RG13 could affect the position of the all important oxyanion hole, involving A237, by perhaps 0.1-0.3Å which will both have an effect on Km and kcat (as the substrate needs to place its carbonyl oxygen in this site and also it needs to be primed for ~2 steps during catalysis (each only
5-fold so changes need to be small). The superpositions of the maltose-free and maltose- bound MBP structures indicate that this β3-229 TEM-1 anchor point region shifts about
2Å near residue 316 in the MBP domain of RG13. Remarkably, another TEM-1 MBP fusion protein has previously been obtained that uses the same MBP fusion site of residue
316 to also obtain maltose-dependent signaling (Guntas et al., 2005). But instead of being inserted in TEM-1 residue 229, this other MBP-TEM-1 fusion protein inserted its MBP domain in TEM-1 residue N170. This suggests that this MBP region has unique and robust features that can transmit maltose-dependent conformational changes to another protein in different ways. This other MBP-TEM-1 maltose-regulated fusion construct was shown to have its maltose-dependent abilities to be negatively affected by increasing the linker length by one and two additional residues (Guntas et al., 2005). This agrees with our RG13 structure-based allostery hypothesis as the linker length and amount of slack are hypothesized to be key for proper maltose signaling in RG13. Note also that in RG13, two linker residues near the second fusion site have been replaced by a single serine residue (Figure 7.1), perhaps as a need to have the proper amount of slack for maltose signaling.
- 148 - We note that TEM-1 residue E166 was previously suggested to be functionally perturbed upon maltose binding but we currently have no structural evidence for a role for this residue. The RG13 NMR data for E166 in the HSQC-TROSY when maltose was present was however already quite weak (Wright et al., 2010) making the absence of this
NMR peak without maltose potentially not as significant. We do however not rule out an indirect effect on the conformation of E166 via β3 strand changes such this strand interacts with the E166-containing Ω loop. Such a conformational change could be similar to the ligand dependent Ω loop disorder we recently observed in variants of a related β-lactamase (Sampson et al., 2011) although such a large Ω loop displacement in
RG13 was however not observed by NMR (Wright et al., 2010).
7.4.2 General applicability of MBP as a sensory allosteric domain in fusion constructs
It is interesting as to why MBP residue 316 is selected for during the generation of two different maltose-regulated TEM-1 fusion constructs as residue 316 is not near where the largest conformational differences are observed upon maltose binding that include a 35° ‘pacman’-type conformational change. Residue 316 is actually situated on the backside of the hinge region of the bi-lobal MBP structure. However, one has to consider as to how these allosteric fusion proteins are generated as the TEM-1 domain is inserted into a single site of MBP, or sites that are in very close proximity (i.e. 316 and
319 of MBP in RG13, (Figure 7.1). Although the linker anchor points of MBP are not where there are the largest differences in distance between an uncomplexed and complexed MBP are found, the anchor points are however near where the largest
- 149 - differences are when one only considers Cα positions of a few residues up- or downstream from each other in the continuous polypeptide chain to allow for insertion of the non-homologous TEM-1 gene at a single site. By calculating Cα-Cα Δdistance measurement of residues i and i + x via substracting the distances from the maltose- bound from the maltose-free MBP structure, the MBP region near 312 lights up as to having the largest Cα-Cα difference for i and i+3 and i and i+6 (Figure 7.10). These calculations suggest that the 312-318 MBP region has the largest shifts, thereby explaining why insertions near residue 316 are capable of transmitting maltose signals to
TEM-1 as is also evident structurally (Figure 7.9A). An additional potential benefit of this region, being on the backside of MBP, is that these regions could exert a lever/torque effect as the fusion points are close to the hinge region. As such, thus region is thus perhaps able to exert a larger force on the movements near its rotation point which probably might be needed as the β3 TEM-1 strand is well lodged into the active site. It is remarkable that the forced evolutionary pressure and double strand nicking in both MBP and TEM-1 resulted in this successful RG13 of having indeed found the MBP 312-318 region and the 230 β3-strand region of TEM-1 indicating that this approach works to find this narrow signaling window region being almost a ‘needle in a hay stack’.
The circular permutation of TEM-1 was thought to have a possible effect on maltose signaling as a different composition linker with different linker length in a related circularly permutated TEM-1/MBP fusion protein called MBP317-347 (GSGGG) had an effect on maltose switching(Liang et al., 2007). This can potentially be explained as these two TEM-1 N-terminal and C-terminal helices that are now linked via this linker
- 150 - are in close proximity to the RG13 cross-over linker as well as β4 strand via interactions involving residues N276 and R244 (see Figure 7.12). Shortening this linker might affect the position and angle between these helices thus potentially affecting the positions of these important interacting regions.
7.4.3 Serendipitous zinc binding and regulation in RG13
RG13 was engineered and selected for to be maltose regulated yet its observed serendipitous zinc inhibition was unexpected (Liang et al., 2007). The crystallographically observed major zinc site in RG13 contains residues H468, D164, and
E477. The double mutant D164A/H468A decreased zinc inhibition about 3 fold suggesting that one or more of these residues have some importance in zinc inhibition. It was previously found that zinc inhibition of RG13 was decreased 30-fold by the
H26A/H287A mutations (Liang et al., 2007). We hypothesize that within RG13, there are multiple possibilities of binding a single zinc ion (our RG13 structural studies even found a second zinc binding site) as other residues could potentially take over the bi-domain zinc binding via rotation and repositioning of the MBP and TEM- 1domains(Liang et al.,
2007). These H26/H287 histidine residues are at the original N-terminal and C-terminal helices of TEM-1, respectively (Figure 7.12). Shifts in these helices can potentially affect maltose signaling in a similar manner as a different linker length and composition between these helices affected maltose signaling in a related MBP-TEM-1 fusion protein
(as mentioned above). Our structural studies do not find any evidence of zinc binding to
H26/H287 in both the P1 and C2 space groups despite these residues being in relative
- 151 - close proximity. The apparent disconnect between the mutagenesis data and the RG13 crystal structure regarding the high affinity zinc inhibition site suggests that H26 and
H287 are part of the high affinity (low) μM zinc inhibition site whereas the RG13 crystal structure, with crystals grown in the presence of 2.5mM zinc, is likely that of a different trapped zinc-inhibited structure due to the higher zinc concentrations used during crystallization. It is not uncommon to have two mutually exclusive zinc binding sites with different affinities which was observed previously in the engineered zinc biosensor that was also due to the higher zinc concentrations used during crystallization (Telmer,
Shilton, 2005).
We note that μM Zn binding can be readily engineered into proteins via juxtapositioning of at least 2 His residues as was achieved in trypsin via surface residues that were flexible in the absence of Zn (Higaki et al., 1990), in kinesin (Greene et al.,
2008), by introducing His residues in loops in CXCR1 (Suetomi et al., 2002), and in retinal binding protein (Schmidt et al., 1996). The remarkable ease of generating a Zn binding site possibly explains why fusion proteins like RG13 can be so relatively easily serendipitously negatively regulated by Zn as RG13. The RG13 construct positions two unrelated domains in close proximity via linkers whose length, position, and conformation, are absolutely critical for allosteric regulation. All RG13 for example would need to do is rotate and perhaps shift the two domains somewhat such that the
His/Asp/Glu and/or other zinc liganding residues are in close proximity locking the RG13 in a fixed conformation thus negating the effects of the positive allosteric compound, maltose. It is interesting to note that the pI of RG13 is 5.53 due to 40 D and 47 E residues. In addition, there are 9 H residues thus having in total 96 residues that can
- 152 - readily form zinc ligands which is 15% of the total RG13 protein (i.e. 637 residues).
Since most of these residues are at the surface, the relative amount of zinc binding residues at the surface is thus even higher than 15% thus having ample opportunities to generate zinc binding sites (Figure 7.11) potentially with just only 2-3 residues, one or two from MBP and one or two from TEM-1 thereby affecting the orientation of the two domains and thus the linker positions, and thus the activity. We speculate that since the
TEM-1 and MBP domains are in a different orientation due to the higher zinc concentration, the H26/H287 residues cannot reach a putative third zinc ligand that would be sufficient to bind zinc and lock the two domains in a different, yet still inhibitory conformation.
In summary of the above discussion, we proposed the RG13 heterotropic allosteric regulation mechanism in Figure 7.13 involving its activation by maltose and inhibition by zinc. It is interesting to note that the maltose and carbenicillin binding sites communicate in both directions as maltose was found to positively influence carbenicillin binding and carbenicillin binding positively influences maltose binding (Guntas et al.,
2004). The different conformations of the subdomains of MBP in the P1 and C2 space groups point to that RG13 is potentially regulated by conformational mobility. This concept entails that a flexible allosteric protein adopts a range of populations of different conformations and the effector binds and effectively redistributes the population of conformations and thereby affects the distant active site (Goodey, Benkovic, 2008). On an aside, to potentially alleviate the potential negative consequences of zinc inhibition,
RG13-like constructs could be further engineered by having a number of surface
His/Asp/Glu and other potential zinc-liganding residues mutated.
- 153 -
Figure 7.1. Overal structure of RG13. (A) RG13 primary structure domain organization.
The TEM-1 domain is circularly permutated, via a GSGGG linker now connecting the original N- and C-termini, which resulted in new N- and C-termini at residues 229 and
228, respectively. This altered TEM-1 is inserted into MBP after residue 316 with MBP residues 317-318 replaced by a single serine residue. (B) Schematic diagram of RG13 crystal structure with zinc bound. TEM-1 and MBP domains are colored magenta and yellow, respectively. Zinc ions are shown as grey spheres and the engineered GSGGG linker is colored cyan. To indicate the position of the TEM-1 active, its catalytic serine residue is highlighted in spheres; (C) Superposition of the two RG13 crystal forms of the
TEM-1 domain reveals a slight different orientation of the MBP domain after TEM-1 domain superpositioning.
- 154 -
Figure 7.2. Active site and linker regions of RG13 (A) 2Fo-Fc density map contoured at
1.0σ depicting the TEM-1 active site region; (B) 2Fo-Fc density maps contoured at 1.0σ for residues 315-330 (the cross-over loop) and (C) residues 571-587 (the re-entry loop);
(D) 2Fo-Fc density map contoured at 1.0σ for residues 377-381 (the engineered GSGGG linker); (E) Temperature representation of the RG13 structure in the P1 space group. The thicker and the more red the backbone is, the more flexible it is due to its higher refined temperature factors.
- 155 -
Figure 7.3. Zinc binding sites in RG13. (A) The main zinc ion binding site in space group P1 bridges MPB domain and TEM domains of RG13 via residues D164 and H468;
(B) main zinc ion RG13 binding site but now for space group C2 showing an additional zinc liganding residue E477. (C) The second zinc binding site in the P1 space group involving H509 and H514 in the TEM domain of RG13 and Y17’ and H39’ of a crystallographically related RG13-MBP domain; (D) Second zinc binding site in the C2 space group structure involve only residues of H509 and H514. The 2Fo-Fc density maps were contoured at 1.5σ and colored blue for the amino acid residues of RG13 and the water molecules. Omit Fo-Fc maps were contoured at 10σ and colored green for the Zn ions. Domain coloring is same as in Figure 7.1 with the crystallographic related neighboring molecules depicted in grey. Water molecules and zinc ions are shown as red and grey spheres, respectively.
- 156 -
Figure 7.4. Structural analysis of the MBP subdomain angle variations. Superposition of subdomain 1 of MBP reveals different domain closure angles of subdomain 2. The superposed structures include uncomplexed MBP structure (PDB ID: 1OMP, grey),
RG13 structure of P1 space group (yellow), RG13 structure of C2 space group (wheat), and maltotriose complexed MBP structure (PDB ID: 3MBP, green). Subdomain 1 of
MBP is defined by residues 1-109 and 261-316.
- 157 -
Figure 7.5. Electron density map showing active site β3 strand displacement in TEM-1 domain of RG13. Superposition of TEM-1 structure (PDB ID: 1ZG4, grey) with active site of TEM domain of RG13 space group P1 (purple). 2Fo-Fc density map contoured at
1.0σ (blue) shows no density observed at the position where β3-strand is originally located in the wt TEM-1 structure. TEM-1 residue numbering are in parenthesis and italicized. The dotted lines represent hydrogen-bond network of RG13 active site via displaced residue R328(241) revealing partial blockage of the active site by the displaced stretch of residues.
- 158 -
- 159 - Figure 7.6. Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure. Superpositioning of wt TEM-1 structure onto the TEM domain in RG13 reveals displacement of β3 strand and part of β4 strand (inset is a zoomed in view with secondary structure elements in transparent representation). The TEM-1 β4 strand is mostly intact yet starts diverging at position R244. The α10 TEM-1 helix which is observed to be more extended in RG13 is labeled. Also labeled and underlined are the MBP helices α14 and
α15 that form part of the linker anchor points in the RG13 fusion protein. TEM-1 β3 strand residue positions K234, S235, A237, and β4 strand residue K244 are indicated as well as the new position of A237 with the RG13 sequence A325. The zinc ion is indicated via a grey sphere.
- 160 -
- 161 - Figure 7.7. R.m.s.d. of backbone atoms calculated relative to the starting structure for
RG13 and TEM-1 molecular dynamics simulations. (A) RG13 in complex with zinc; (B)
RG13 with zinc omitted from model; (C) wt TEM-1 molecular dynamics simulation. All simulation were carried out for 17ns.
- 162 -
Figure 7.8. Molecular dynamics simulations reveals zinc dependent changes in relative orientation of MBP and TEM-1 domains. MBP and TEM domain of our RG13 crystal structure was colored yellow and purple respectively. The GSGGG linker connecting the
N-and C-terminal of TEM-1 was colored cyan. The final coordinates of the 17ns zinc-
RG13-simulation is colored blue, the no-zinc-RG13 simulation is green, and the wt TEM-
1-simulation is colored gray. A TEM-1 active site inhibitor was included in orange spheres to depict the location of the TEM-1 active site.
- 163 -
Figure 7.9. Modeling of RG13 fusion protein with a catalytically competent TEM-1 domain near MBP fusion site to relay maltose-induced conformational changes. (A)
Superpositioning of the subdomain 1 of MBP (residues 1-109 and 261-316) from maltose-free MBP (red, PDBid 1OMP), maltotriose-bound MBP (green, PDBid 3MBP), and RG13 P1 space group structure (yellow). MBP anchor/fusion points labeled ‘1’
(R316) and ‘2’ (A586(319)) to which TEM-1 is fused are labeled and colored as orange and red spheres, respectively. The respective MBP helices to which these two MBP anchor points are adjacent to are helices α14 and α15, respecively. The positions of S585
, introduced as part of constructing RG13 (Figure 1A), and the TEM-1 residues 228, 229, and 230 in the RG13 fusion protein are labeled; the other connecting TEM-1 residues are shown as dashed grey lines. Shifts in the Cα positions of the α15 helix going from unbound to maltotriose bound MBP conformation is indicated by dashed arrows. (B)
Close-up view of RG13 fusion site showing the MBP (yellow) and TEM-1 domains
(magenta) of RG13, and the modeled position of an intact TEM-1 domain (blue, PDBid
1ERO which includes a boronic acid inhibitor to indicate the position of the active site).
To position the wt TEM-1 structure while avoiding steric clashes with MBP, TEM-1 residue F230 (green sphere) and equivalent RG13 residue F318 were superpositioned during this modeling so that TEM-1 could be oriented such that its second anchor/fusion
- 164 - point, G228 (green sphere), is in close proximity to its equivalent G584 residue in RG13
(magenta sphere). These TEM-1 anchor/fusion positions in RG13, F318 and G584, are at a distance that is larger than found within the wt TEM-1 structure (residues F230 and
G228, respectively) and a dashed arrow shows the movement that is needed for G584 in
RG13 to reach the G228 position as found in wt TEM-1. The MBP anchor point helices
α14 and α15, which move relative to each other upon maltose/maltotriose binding, are shown in yellow helix cartoon representation. The movements anticipated to occur in (A) and (B) are in roughly the same direction as shown by their respective dashed arrows suggesting that maltose binding could bring TEM-1 residues G228 and F230 to a shorter more native distance with residues G228 being the start of the important active site β3 strand. TEM-1 β3 and β4 strands are labeled; (C) zoomed out view as in (B). TEM-1 bound boronic acid inhibitor (BAI) is shown in magenta stick representation to pinpoint the position of the TEM-1 active site. The black line and small black helix depict the
RG13 residues that normally form the TEM-1 β3 and part of β4 strand yet now form the linker between the TEM-1 and MBP domains in the zinc inhibited RG13 structure. The rectangular dashed box in RG13 highlights the position of where the β3 strand used to be in an intact TEM-1 conformation.
- 165 -
Figure 7.10. The Cα-Cα difference plot for maltose-free and maltose-bound MBP. Cα-
Cα distance differences for i=i+3 (black) and i=i+6 (grey) of the maltose-free MBP
(1OMP) and maltotriose (3MBP) structures are plotted per residue. The plot highlights where the largest maltose/maltotriose-dependent (local) Cα shifts occur of nearby residues that are either 3 or 6 residues further in the sequence. Arrow depicts where largest consistent shifts occur (near MBP residue 312).
- 166 -
Figure 7.11. Surface depiction of RG13 showing potential zinc liganding surface residues. Histidines, aspartates, glutamates, asparagines, glutamines, tyrosines, and cysteine residues are color coded as indicated. The RG13 molecule is in surface representation and the main zinc ion is shown as a grey spheres.
- 167 -
- 168 - Figure 7.12. Regulatory zinc binding sites in RG13. RG13 (yellow and magenta) is superimposed onto wt TEM-1 (in grey). The crystallographically observed zinc binding ligands D164, H468, and E477 in RG13 are shown in ball-and-stick. Active site residues
S70, E166, R244, and N276 are also shown. MBP is depicted in yellow with its anchor/fusion point helices α14 and α15 shown as yellow transparent rods. TEM-1 is shown in magenta with its original N- and C-terminal helices α1 and α12 shown in magenta transparent rods. The engineered GSGGG linker between these helices is shown in blue. The position of the original TEM-1 β3 strand position is indicated by the transparent red strand. The cross-over loop and re-entry loops between MBP and TEM-1 are labeled 1 and 2, respectively. The positions of the RG13 residues previously shown to be involved in the zinc-mediated inhibition of RG13, H375 and H382 (i.e. TEM-1 residues 26 and 289), are shown in ball and stick and labeled. TEM-1 helix α10 is also labeled due to its close proximity to the active site, close proximity to TEM-1 α12, and observed structural differences between RG13 and TEM-1.
- 169 -
- 170 - Figure 7.13. Schematic diagram of heterotropic allosteric regulation of RG13 by maltose and zinc. MBP (yellow) and TEM-1 (magenta) are shown in cartoon representation. The
5-stranded β-sheet in TEM-1 is depicted including the β3 and β4 strands; the TEM-1 active site location is highlighted by an ‘X’. The MBP anchor/fusion point helices α14 and α15 are depicted with its fusion points colored orange and red, respectively. RG13 in the unbound state (left lower corner) is drawn such that the N-terminal part of the β3 strand is not in its original TEM-1 position (original position shown in transparent) as indicated by NMR studies. This β3 strand is speculated to undergo a conformational change upon maltose binding such that β3 strand is relaxed enough to more completely adopt a native like β-strand conformation in the active site. This relaxing of the N- terminal part of the β3 is thought to occur due to shortening of the distance between the anchor/fusion points in MBP to which the N-terminus of this β3 strand is attached. The non-competitive inhibition by zinc (grey sphere) of RG13 occurs by providing an additional contact point between MBP and TEM-1 thereby twisting and altering the orientation between these two domains such that the linker region requires additional slack and thereby pulls the β3 strand region out of the TEM-1 active site. Inset upper right: individual structures of (unfused) wt TEM-1 and MBP for comparison.
- 171 - Table 7.1. Data collection and refinement statistics
Data collection space group P1 C2 cell dimensions a, b, c (Å) 48.32, 74.18, 103.13 124.12, 47.78, 107.87 α, β, γ (deg) 83.54, 77.64, 89.98 90.00, 114.17, 90.00 wavelength (Å) 1.0810 1.0810 resolution (Å) a 50.00-2.30 (2.38-2.30) 50.00-2.30 (2.38-2.30) Rsym 5.4 (22.3) 5.1 (32.8) I/σI 12.6 (3.8) 15.4 (2.7) Completeness (%) 95.9 (95.5) 88.0 (88.2) Redundancy 1.9 (1.9) 2.3 (2.3) Refinement Resolution range (Å) 31.45-2.29 (2.294-2.354) 42.32-2.30 (2.360-2.301) no. of reflections 56130 22012 Rwork/Rfree 23.2/29.2 23.3/29.1 no. of atoms: protein, zinc ion, water 9851, 4, 490 4830, 2, 134 rmsd b bond length (Å) 0.007 0.006 bond angles (deg) 0.960 0.942 average B-factors (Å2) Protein, zinc ion, water 34.385, 31.573, 34.750 41.047, 40.825, 36.184 Ramanchandran plot (%) core regions 91.7 92.1 additional allowed 8.1 7.5 generously allowed 0.2 0.2 disallowed regions 0.0 0.2 a Numbers in parentheses refer to the highest resolution shell. b rmsd, root-mean-square deviation
- 172 - Table 7.2. Kinetics data from the literature (Liang et al., 2007;Guntas et al., 2005)
-1 -1 kcat (s ) Km (μM) kcat/Km (s / μM)
RG13 200 ± 40 550 ± 120 0.36
RG13+maltose 620 ± 60 68 ± 4 9.12
TEM-1 900 110 8.18
RG13+zinc Ki = 2.7 ± 0.1 μM
- 173 - CHAPTER 8
Summary and Future Directions
8.1 Summary
Expression of β-lactamases is the most common resistance mechanism against β- lactams. The number and types of β-lactamases are growing rapidly. As a consequence, new types of β-lactam antibiotics are developed and marketed, which includes five generations of cephalosporins and carbapenems. Moreover, a combination therapy was developed with a β-lactamase inhibitor co-administrated with an antibiotic. The current three β-lactamase inhibitors used clinically (Clavulanic Acid, Sulbactam, and
Tazobactam) are Class A β-lactamase inhibitors, which have little or no effectiveness for
Class B, C and D β-lactamases. In a most recent study, a triple combination was investigated. This triple combination comprised a siderophore monobactam, a novel bridged monobactam and clavulanic acid and demonstrated activity against all the four classes of β-lactamases (Page et al., 2011). However, the fundamental urgent need is still to develop more potent β-lactam antibiotics or inhibitors that harbore a broader spectrum.
For this purpose, we need to understand the structural basis of β-lactamases overcoming
β-lactam antibiotics and inhibitors and we need to exploit this knowledge to develop improved β-lactam antibiotics or inhibitors, which are the focus of this thesis work. In addition, in this thesis work we also exploit another way of inhibition, which is through heterotropic allosteric regulation.
- 174 - In this thesis work, we determined the crystal structure of KPC-2 and provide insights into carbapenemase activity of KPC-2, which contributed increased understanding of the carbapenemase resistance. KPC-2 manages to hydrolyze carbapenems by adopting a shallower active site with a flexible catalytic residue S70
(chapter 5). In addition, we exploited three strategies for inhibitor development: (1) exploited R1 group intra-molecular chemistry to form suicide inhibitors; (2) exploited trans-enamine stabilizing inhibitor via R2 group; and (3) transition state stabilization via boronic acid transition state inhibitors (Figure 8.1A). The current three inhibitors clinical used contains no R1 group and a relatively small R2 substituents (Figure 8.1B).
Tazobactam and sulbactam differ from clavulanic acid in that the former are penam sulfones. The sulfone group of tazobactam and sulbactam are proved to be a better leaving group. Also the sulfone group is involved in forming an intra-molecular hydrogen bond that helps stabilizing the trans-enamine intermediate (Padayatti et al.,
2004;Padayatti et al., 2005).
To improve the potency of β-lactamase inhibitors, one approach is to stabilize and trap one of the intermediate along the reaction pathway within bacterial regeneration cycle. In this study, we selected SA1-204 (provided by Dr. Buynak) and penem 1
(provided by Wyeth) to study the R1 group intra-molecular chemistry to form suicide inhibitors. Both of these two compounds contain a methylidene heterocycle substitution at C6 position (R1 group), but they follow distinct reaction pathways (chapter 3). SA1-
204 acylates catalytic S70 forming a bi-cyclic ring structure, while penem 1 acylates catalytic S70 forming a 7-membered-thiazeping-ring structure. We determined crystal
- 175 - structures of these two compounds with SHV-1 β-lactamase revealing that both compounds are resistant to deacylation due to the conjugation effect and the resultng decreased electrophilicity of the carbonyl carbon atom. SA1-204 also manages to stabilize the acyl-intermediate by positioning the ester carbonyl out of the oxyanion hole.
Next, we exploited R2 group interactions to stabilize the trans-enamine species.
SA2-13 was rationally designed by our group and its inhibition efficacy against SHV-1 β- lactamase was tested (Padayatti et al., 2006). The design strategy was to substitute the triazolyl moiety of tazobactam with a negatively charged carboxylate attached through an appropriately sized linker so that this R2 group could interact with enzyme’s carboxylate binding pocket. Trans-enamines are resistant to hydrolysis due to its being less likely to be attacked by the deacylation water molecule. In this thesis work, to further test this strategy against IR β-lactamase, we determined the crystal structure of SA2-13 in complex with IR S130G SHV variant and found thatSA2-13 to be a very adaptable trans- enamine stabilizing inhibitor against IR SHV β-lactamase (Chapter 4). To improve the cell entry of SA2-13, an analogue PSR3-226 was synthesized by Dr. Buynak’s lab.
PSR3-226 was designed by changing the negative charged carboxyl moiety into a neutral amide moiety to improve the cell entry. Also, the linker length was reduced to test the importance of the linker length. PSR3-226 was demonstrated to have the ability to get into the cell (personal communication Dr. Bonomo). We succeeded in determining the crystal structure of PSR3-226 complexed KPC-2 β-lactamase, which is a clinical very important carbapenemase (Chapter 6). This compound forms a trans-enamine intermediate in the active site of KPC-2. Similar to the penam sulfones of tazobactam and
- 176 - SA2-13, the acyl-intermediate of PSR3-226 has its ester carbonyl positioned in the oxyanion hole, has an intra-molecular hydrogen bond to stabilizing the trans conformation, and has the favorable interactions of the C3 carboxylate group with enzyme active site. However, unfortunately the sulfone group interferes with the wild- type position of W105 of KPC-2 and also the amide linker points toward the bulk solvent and makes no interactions with the enzyme. These insights yield clues regarding the importance of the linker length and improvement for future drug design.
Then we tested the third strategy by utilizing compounds mimicking the transition state. These boronic acid compounds we are interested all contain the boron-hydroxyl head group. The boron atom of these boronic acid compounds can form a covalent bond with the hydroxyl group of the serine residue. Therefore, these boronic acid compounds could approach the catalytic residue S70 of the Class A β-lactamases, covalently attach to
S70 and block the enzyme active site. The geometry around boron atom is tetrahedral and could mimick either the acylation transition state or deacylation transition state. In this thesis work, we studied a cefoperazone BATSI, which was synthesized and provided by
Dr. Prati’s lab. This compound has the R1 side chain attached to the boron-hydroxyl group. We determined the crystal structure of cefoperazone BATSI in complex with
SHV-1 β-lactamase (Chapter 2). Our structure reveals that cefoperazone BATSI mimicks the deacylation transition state as one of the hydroxyl group takes position in the oxyanion hole and the other hydroxyl group takes position of the deacylation water molecule (not the position of the leaving nitrogen atom in the Michaelies-Menten complex), suggesting that there could be differences between the Ki of the BATSIs and
- 177 - the Km of the corresponding substrates. Also, our structure reveals that Y105 was positioned by the phenol group of the inhibitor in an unusual outward position compared with the WT SHV-1 structure. Two analogues were therefore designed and synthesized
(by Dr. Prati’s lab) with the purpose to maintain favorable interactions (ie, with the boron-hydroxyl, the amide part, and the piperazine-containing ring) and the purpose to lessen the impact that the cefoperazone BATSI has on Y105 conformation. And we also determined the crystal structure of these two analogues of cefoperazone BATSI with
SHV-1 β-lactamase (Chapter 2). Both designed BATSIs have improved inhibitory potencies and allow less strain on Y105 (structures reveal alternative conformation for
Y105). And our structure reveals that, for future BATSI improvements, the length of the phenol moiety could be increased and an additional carboxyl moiety could be added to the aromatic ring such that it could reach the conserved carboxylate binding pocket.
Finally, we investigated the mechanism of heterotropic allosteric regulation of an engineered β-lactamase. RG13 was engineered by Dr. Ostermeier’s lab in such a way that the reshuffled gene of TEM-1 β-lactamase was inserted into the gene of MBP. RG13 was demonstrated to be a molecular switch, the β-lactam hydrolytic activity of which was positively regulated by maltose and negatively regulated by zinc ion. As neither maltose nor zinc ion regulates the activity of TEM-1, we are interested in understanding how
RG13 achieves this. In chapter 7, we determined the crystal structure of RG13 in the presence of zinc ion. The structure reveals that zinc in a non-competitive inhibitor as it takes a position differently from the substrate-binding site. The structure also reveals that the zinc inhibits enzyme activity by having a distorted active site with the critical β3-
- 178 - strand of TEM-1 now becoming a short helix followed by a linker connecting to the MBP domain. Combined with molecular dynamics simulation and molecular modeling, we proposed that the linker length (the slack extent of the linker in our case) is important for reforming the functional active site. Without the presence of zinc ion, the additional contact point between TEM domain and MBP domain are removed and allows domain rotation so that the orientation of the two domains is less twisted and likely allows the partially reinsertion of the β3-strand. Moreover, with the binding of maltose, the MBP domain has a ~30 degree bending motion, which brings closer the two helices at the backside of the hinge and allows even more slack of the linker so that the β3-strand could now likely to be fully restored.
In conclusion, our structural characterization of these enzymes and enzyme- inhibitor complexes revealed significant active site chemistry and mechanism of inhibition. Penem 1 has nano-molar inhibition capability and shows extended-spectrum inhibitory characteristics against Class C β-lactamases. SA1-204 forms an extremely stable acyl-intermediate resistant to be hydrolyzed. PSR3-226 has the advantage over
SA2-13 as it has in vivo activity. Although cefoperazone BATSI only have micromolar inhibition constants for the β-lactamases tested, there are BATSIs achieving encouraging nanomolar inhibition constants for TEM-1 (Ness et al., 2000). Our results presented herein could serve as one of the building blocks for the future structure-based drug design. And additional chemical, structural, and functional work is needed to further improve these potential β-lactamase inhibitors.
- 179 - 8.2 Future Directions
SHV-1 and KPC-2 are good model systems to structurally characterize enzyme- inhibitor complexes. However, KPC-2 has proven to be difficult in trapping the inhibitor in the active site. Reasons could be the intrinsic hydrolytic and fast on and off character of KPC-2 since it has broad substrate profile. Or it could be the reason of the buffer pH, since KPC-2 was crystallized at extreme pH either 9.0 or 4.0. To capture additional KPC-
2 complexed structures easier, KPC-2 E166A mutant is a good candidate which demonstrated previously to significantly slow down the rate of deacylation and trap the inhibitor intermediate (Padayatti et al., 2004). I have made the KPC-2 E166A mutant and tested its overexpression and purification (Figure 8.2). About 11mg pure protein could be obtained from 10L LB culture. Now it is at the stage of making diffractive crystals so that we could use it to soak with a variety of inhibitors. Also, KPC-2 S70C/A mutants were made and sequence confirmed which might be helpful for us to capture a Michaelis-
Menton Complex. For elucidation of the allosteric regulation mechanism of RG13, we used the crystal structure in the presence with the negative effecter zinc ions combined with molecular dynamic simulation and molecular modeling techniques. To have a full accurate picture of the heterotropic regulation mechanism, it would be useful to also have crystal structures of apo RG13 and RG13-maltose complex. From a variety crystallization screening, one of the preliminary conditions is shown in Figure 8.3. Further screening and optimization are needed.
- 180 -
A
B
- 181 - C
Figure 8.1. (A) Proposed reaction pathway of an ineffective inhibitor being hydrolyzed based on previous work; (B) Chemical structures of three clinical used β-lactamase inhibitors; (C) Chemical structures of inhibitors that have been discussed in this thesis work. Green dashed circles highlight the R1 substituent. Purple dashed circles highlight the R2 substituent. Compounds with blue circles are boronic acid transition state inhibitors, which all contain the boron-hydroxyl head group.
- 182 -
Figure 8.2. Elution chromatogram of KPC-2 E166A on the Superdex 200 column. The
Peak at 17.49ml represents KPC-2 E166A.
- 183 -
Figure 8.3. Needle clusters of RG13 in the presence of maltose. Reservoir solution contains 2.4 M Ammonium phosphate dibasic, 0.1 M Tris pH 8.5 (SaltRx: #52).
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