Crystal structure of aminoglycoside phosphotransferase(9)-la in complex with ADP and spectinomycin reveals conformational change upon substrate binding

Jiyoung Hwang

Master of Science

Department of Biochemistry McGiII University Montreal, Quebec, Canada

A thesis submitted to McGiII University in partial fulfillment of the requirements of the degree of Master of Science

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Aminoglycosides comprise a class of small molecules that have found their way to being one of the most used antibiotics for fighting bacterial infections. Their effectiveness has, however, been compromised in recent years due to the rise in the number of aminoglycoside-resistant strains of bacteria. Their resistance is conferred by enzymes that modify aminoglycosides that are now ubiquitous in both Gram-positive and Gram-negative bacteria. This has prompted a renewed look towards seeking novel drugs via structure-activity relationships. Aminoglycoside phosphotransferases (APH) phosphorylate hydroxyl moieties present on aminoglycosides that render them ineffective. The majority of APH's exhibits broad substrate specificity. Interestingly, APH(9)-la, isolated from Legionella pneumophila, phosphorylates one compound, spectinomycin. To further establish the basis for designing a universal drug for APH's and the rationale behind its substrate specificity, the crystal structure of APH(9)-la in its ternary form (with bound ADP and spectinomycin) was solved to a resolution of 2.5 A. The overall architecture reveals a helix (aC), characteristically larger than its other APH counterparts, which contains three key residues (Arg284, Asp288, and Tyr292) involved in spectinomycin binding. In addition, there are only two acidic residues that participate in its binding, setting APH(9)-la apart from multi­ aminoglycoside detoxifying enzymes. We propose that these features are responsible for the enzyme's single-substrate profile. Furthermore, the structure reveals a significant lobe movement for substrate binding, previously unseen in this class of enzymes. An inhibition study using eukaryotic protein kinase inhibitor, CKI-7, showed it indeed does inhibit the enzyme (KI =(159 ± 11) !-lM), though with 2.5 times less effectiveness than for APH(3')-lIla. Search for possible alternative substrate for this enzyme showed that APH(9)-la does not phosphorylate substrates of homologous kinases. The enzyme's importance in the bacteria's survival is questionable; however, its structure will still serve to augment our growing knowledge on aminoglycoside modifying enzymes. This information will be key to future novel drug designs.

ii ACKNOWLEDGEMENT

The apo-APH(9)-la structure was solved by Dr. Chris Lemke, a former post­ doctorate fellow in Berghuis Lab. It was solved by the MAD (Multi-wavelength Anomalous Dispersion) method to a resolution of 1.7 A. This structure was used to phase the data for the APH(9)-la ternary complex to 2.5 A resolution by molecular replacement.

1 would like to extend my most sincere appreciation and gratitude to the many people whom 1 have come across throughout my graduate studies. 1 am most indebted to my supervisor, Dr. Albert Berghuis for his great guidance and support. Special thanks are due also to ail the pa st and present members of the Berghuis Lab; Dr. Chris Lemke, Dr. David Burk, Dr. Bing Xiong, Elaine Kwok, Desiree Fong, Noboru Ishiyama, Ahamad Mirza, Magda Korczynska, Nick Skouris, and Oliver Baettig.

Many thanks also to Leonid Flaks and Martin McMilian at X8C NSLS Brookhaven National Laboratories for their assistance during data collection.

Finally, 1 would like to acknowledge the Canadian Institute of Health Research strategie training in Chemical Biology at McGiII University for their funding support.

iii DEDICATION

To my twin sister

iv TABLE OF CONTENTS

DEDICATION ...... iv TABLE OF CONTENTS ...... v LIST OF TABLES ...... vi LIST OF FIGURES ...... vii LIST OF SYMBOLS ...... viii CHAPTER 1: Introduction ...... 1 1.1 Mechanism of Antibiotic Resistance ...... 1 1.2 Aminoglycoside modifying enzymes ...... 2 1.3 Chromosomal origin of APH ...... 4 1.4 Aminoglycosides ...... 6 1.5 Future of Drug Design ...... 11 1.6 Legionella pneumophila ...... 14 1.7 Objective ...... 15 CHAPTER 2: Experimental Procedures ...... 16 2.1 Materials ...... 16 2.2 Protein Purification ...... 16 2.3 APH(9)-la Co-Crystallization with ADP and Spectinomycin ...... 17 2.4 Data Collection and Processing ...... 17 2.5 Structure Determination and RefinemenL ...... 17 2.6 Enzyme Inhibition Kinetics ...... 18 2.7 Substrate Assays ...... 19 CHAPTER 3: Results ...... 22 3.1 Protein Purification ...... 22 3.2 Co-Crystallization ...... 24 3.3 Ternary Structure of APH(9)-la ...... 25 Overall Architecture ...... 25 Aminoglycoside binding ...... 29 Nucleotide binding ...... 35 3.4 Inhibition Studies ...... 37 3.5 Substrate Assays ...... 39 CHAPTER 4: Discussion ...... 40 4.1 Comparison with APH(3')-lIla ...... 40 4.2 Conformational Change ...... 48 4.3 Comparison with Choline Kinase ...... 57 4.4 Enzyme Inhibition by CKI-7 and Substrate Assays ...... 59 CHAPTER 5: Conclusion ...... 62 APPENDIX ...... 63 A. Enzyme Kinetics ...... 63 B. Monitoring APH(9)-la reaction ...... 64 C. Cryo-crystallography ...... 65 LIST OF ABBREVIATIONS ...... 67 REFERENCES ...... 68

v LIST OF TABLES

Table 1: Data collection and refinement statistics ...... 20

vi LIST OF FIGURES

Figure 1: Chemical structures of aminoglycosides ...... 7 Figure 2: Structures of semi-synthetic aminoglycosides ...... 9 Figure 3: Eukaryotic protein kinase inhibitors, CKI-7 and CKI-8 ...... 13 Figure 4: APH(9)-la Purification ...... 23 Figure 5: Micrograph of APH(9)-la co-crystallized with spectinomycin and ADP ...... 25 Figure 6: Ove rail architecture of APH(9)-la in complex with ADP and spectinomycin ...... 26 Figure 7: Sectional representation of APH(9)-la ...... 27 Figure 8: Observed electron densities of ADP and spectinomycin in a refined 2Fo-Fc map contoured at 1.00...... 30 Figure 9: Spectinomycin binding site of APH(9)-la ...... 31 Figure 10: Schematic representation of hydrogen bond interaction between spectinomycin and APH(9)-la ...... 32 Figure 11: Interactions involving the conserved phosphotransferase motif, HxDxxxN ...... 34 Figure 12: ADP binding ...... 36 Figure 13: APH(9)-la Inhibition by CKI-7 ...... 38 Figure 14: Alternative Substrate Assay ...... 39 Figure 15: Sequence alignment of APH(9)-la from Legionella pneumophila with APH(3')-lIla from Enterococcus faecalis (PDB 1J7L), Choline kinase-2A from Caenorhabditis elegans (CKA-2; PDB 1 NW1), and cAMP-dependent kinase from Mus musculus (cAPK; PDB 1 BKX) ...... 43 Figure 16: Ternary complex of APH(3')-lIla (PDB 1L8T) ...... 52 Figure 17: Ribbon representation of superimposed APH(9)-la structures ...... 53 Figure 18: Magnified picture of spectinomycin binding site ...... 54 Figure 19: Magnified picture of superimposed APH(9)-la nucleotide binding site ...... 55 Figure 20: Sectional view of (a) APH(9)-la and (b) Choline Kinase-2A (PDB 1 NW1) ...... 58 Figure 21: Chemical structures of choline and ethanolamine ...... 61 Figure 22: Pyruvate kinase and lactate dehydrogenase-coupled reactions used for APH(9)-la assay ...... 64

vii LIST OF SYMBOLS

Km Michaelis-Menton constant

KI Inhibition constant

V max Maximum velocity

kcat rate constant A Angstrom

Et Total enzyme S Substrate

viii CHAPTER 1: Introduction

Historically, antibacterial drugs have garnered much attention for their important applications in the treatment of microbial infections. Aminoglycosides that include the likes of streptomycin and kanamycin, and ~-Iactams that include penicillin and its derivative, methicillin, have long been mainstays in the antibacterial drug market and thus have enjoyed great usage world wide. Their first discoveries, the former in 1944 by Selman Waksman and the latter in 1928 by Alexander Fleming, were welcomed with much excitement and promise, rapidly vanquishing bacterial infections such as tuberculosis that had plagued the medical community much of the early 20th century. However, with that followed the inevitable overuse of these drugs, popularized by their great effectiveness, ease of use, and ready availability. Soon after, antibiotics, so often regarded as their 'magic bullet', began to lose its magic, succumbing to resistant bacteria. Little was known about the survival mechanisms of bacteria then, and the concept of gene mutations and exchanges was not realized until antibiotic­ resistant strains of bacteria tirst began to show their presence. The unexpected rapid dissemination of these survival genes that conferred their resistance left the scientific community scrambling to find new ways to combat the growing problem of antibiotic resistance. What is disconcerting is that the list of resistant strains of bacteria continues to grow and shows no signs of slowing down.

1.1 Mechanisms of Aminoglycoside Resistance

Through selective pressures from the environ ment, bacteria have developed several mechanisms to circumvent the effects of aminoglycoside. Four main aminoglycoside resistance mechanisms have been proposed; impaired drug uptake, enhanced efflux pump action, alteration of antibiotic target, and enzymatic modification. Diminished aminolgycoside uptake was tirst reported in Pseudomonas aeruginosa. Its precise mechanism of action is still poorly understood but defective porin channel is believed to be one of the factors [1] .

1 The second mechanism of increased efflux pump action [2, 3] promotes the rapid removal of intracellular aminoglycosides via specific pump systems. They have been reported in several pathogenic bacteria including E. coli [4], Burkholderia pseudomallei [5] and P. aeruginosa, contributing to aminolgycoside impermeability-type resistance [6-8]. Modifications of antibiotic target by gene mutations and methylations have also been found to be effective at conferring resistance. For example, a mutation on the 16S rRNA has been reported to cause spectinomycin resistance in Escherichia coli [9], and clinical isolates of Neisseria meningitidis and Neisseria gonorrhoeae [10]. Reports of resistance by enzymatic methylation of 16S rRNA is generally confined to aminolgycoside­ producing actinomycetes such as Streptomyces spp. and Micromonospora spp. This mechanism had not been reported for bacteria of clinical relevance until recently when 16S rRNA methylase was identified as the cul prit behind aminoglycoside resistance in Pseudomonas aeruginosa [11], Serratia morcescens S-95 [12] in Japan, and Klebsiella pneumoniae in France [13]. The fourth mechanism of enzymatic modification of aminoglycosides is by far the most prevalent strategy employed by bacteria to counter their effects; therefore, these enzymes are the most important and interesting targets to focus on for novel drug designs.

1.2 Aminoglycoside modifying enzymes

Aminoglycoside modifying enzymes are classified into three distinct categories based on the type of modification they confer on the aminoglycoside: 0- phosphotransferase (APH), O-nucleotidyltransferase (ANT), N-acetyltransferase (ACC). The classification and nomenclature of these enzymes have been reviewed with extensive detail by Shaw et al [14], but briefly the nomenclature is as follows; the number in brackets following the abbreviated enzyme name denotes the site of modification, the roman numeral following indicates its resistance profile, and finally, the small-case letter designates a unique gene. Amongst these three types of enzymes, their catalytic efficiencies and the

2 amount of enzyme present within an organism varies greatly. It has, however, been shown that phosphotransferases provide the highest level of resistance amongst these enzymes [15]. There are twenty-one reported APH enzymes, of which four have been kinetically characterized [16-19]. The efficiency of 0- phosphotransferases is unquestionable, with the kcat/Km values ranging from 106 8 1 to the diffusion limit of 10 M- S-1 [19-21]. In addition, most APH's have a broad substrate spectrum with the ability to detoxify multiple aminoglycosides. The resulting modified aminoglycosides were shown to exhibit as much as 2000-fold reduced binding affinities to its target, the ribosomes [22].

APH(3')-llla is the most well-studied enzyme in this category. Its crystal structures in apo- (no ligands), holo- (with bound nucleotide, ADP or AMPPNP), and ternary forms (with bound nucleotide and aminoglycoside) have already been determined [23-25]. The existing enzyme kinetics and structural studies suggest that this enzyme carries out its function via an ordered bi-substrate reaction called the Theorell-Chance mechanism [18, 21, 26-30]. In this mechanism, the ATP binds first to the enzyme, followed by the aminoglycoside. The y-phosphate from ATP is then transferred to a specifie hydroxyl group on the antibiotic, and the phosphorylated product is released. The reaction is completed with the rate-limiting release of ADP from the enzyme. Substrate inhibition kinetics, solvent isotope effect, and thio-effect ail lend support to this proposed mechanism. Site-directed mutagenesis of APH(3')-lIla allowed the identification of aspartate-190 and lysine-44 as being critical for catalysis. Similarly, APH(9)­ la, also known as spectinomycin kinase, isolated from Legionella pneumophila was recently characterized and was shown to carry out its activity using the same mechanism with two residues mirroring those of APH(3')-lIla (Lys52 and Asp 212) being critical for catalysis. These studies introduced the question of how these specifie residues mediated the reaction. It has been speculated that the aspartate residue functions as the general base that abstracts the proton off a specifie hydroxyl group of an aminolgycoside, which then participates in subsequent nucleophilic attack of the y-phosphate of ATP. However, the exact

3 role of this putative base remains in a cloud of speculation, for an alternative role has been suggested [31, 32]. Recent studies of kinase provide support for the aspartate acting as a hydrogen bond acceptor that consequently orients the hydroxyl group of the substrate for subsequent nucleophilic attack, and there now seems to be a general consensus that hydrogen bonding and not deprotonation is the primary role ofthis residue [31-34]. This invariant aspartic acid residue is, in fact, conserved across ail APH enzymes and eukaryotic protein kinases as part of the Brenner's phosphotransferase motif, Y/HxDxxxxN (x corresponds to any amine acid) [35] .

1.3 Chromosomal origin of APH

A large percentage of aminoglycoside modifying enzyme genes exists as part of a plasmid or transposable element [36]. This property facilitates the rapid dissemination of antibacterial genes via lateral gene transfers within and across species, which explains the ubiquitous nature of these enzymes in both Gram­ negative and Gram-positive bacteria. Interestingly, many aminoglycoside modifying enzyme genes have been found as part of organisms' chromosomes. It is understandable that aminolgycoside-producing bacteria should have these genes for its intrinsic resistance; however, sorne suggestions of these enzymes taking on additional physiological roles have surfaced for those that do not produce these compounds [19, 37]. A new aminoglycoside phosphotransferase, APH(9)-la, also known as spectinomycin kinase, that was recently isolated from the chromosomes of Legionella pneumophila [37] has been under this speculation [19]. APH(9)-la is one of only a handful of enzymes that is known to phosphorylate and detoxify spectinomycin. It phosphorylates the hydroxyl group at the 9-position of spectinomycin (Fig. 1). The position of phosphorylation was corroborated by rigorous NMR and mass spectroscopy studies [19]. Ali other spectinomycin detoxifying enzymes identified thus far have been of Gram­ positive bacterial origin and ail have been nucleotidyltransferases. A distinguishing feature of APH(9)-la is that it operates exclusively on

4 spectinomycin. This is rare for APH class of enzymes whose majority exhibit broad substrate specificity, with the capacity to phosphorylate multiple aminoglycosides. More recently, an additional spectinomycin kinase was identified from Streptomyces f1avopersicus [38]. It is believed to be part of the spectinomycin biosynthesis gene cluster. The difference between the function of APH(9) in these two organisms lies in the fact that Legionella spp. is not known to biosynthesize spectinomycin, whereas Streptomyces spp. is the primary producer of this antibiotic. The aph9 gene in the latter is explained by the organisms' necessity to confer resistance to its own biosynthesized molecule. However, that aph9 gene should reside on a chromosome poses some questions on its role in the organism's life. Legionella's natural niche may require the organism to have spectinomycin resistance, but this has not been firmly established. It was thought that the genetic environment where aph9 gene resides may suggest an alternative physiological function of APH(9)-la. The genome sequencing of Legionella pneumophila was recently completed [39]. Inspection of the local aph9 gene environment, however, tells little about its physiological role. The gene has no preceding -10 and -35 promoter sequence, which was explained by a suggestion that perhaps it is part of an operon [37]; however, sequencing information shows that its surrounding genes' functions have great variability ranging from aminopeptidase to adenylate cyclase. These two paradoxical findings provide indeterminate information, leaving aph9's possible physiological role difficult to pinpoint, and at best ambiguous. The gene could otherwise be an evolutionary remnant that has lost its function through time. Nevertheless, other chromosomally encoded aminoglycoside-resistant genes have been implicated in taking on additional roles. Gentamicin 2'-N­ Acetyltransferase (AAC(2'» from Providencia stuartii was shown to acetylate both peptidoglycan and aminoglycoside, leading to the speculation that this enzyme is involved in the biosynthesis of cell membranes [40]. The possible function of AAC(2')-lc isolated from M.tuberculosis was also probed using crystal structures with bound substrates. From the specific interactions between the residues and the substrates, the enzyme was suggested to be involved in the

5 biosynthesis of mycothiols. This alternative function explains why the organism does not use AAC(2')-lc to confer resistance to aminoglycosides [41]. Interestingly, aph(3')-lIb from Pseudomonas aeruginosa was found to be part of the hydroxylase Hpa operon. In the presence of HPA (4-hydroxyphenylacetic acid), a carbon source for the bacteria, a heightened resistance to neomycin was conferred to the bacteria [42]. Such correlation between aminoglycoside resistance and metabolic pathways has not been reported for other chromosomal phosphotransferase enzymes. The existence of chromosomally-encoded modifying enzymes entertains compelling hypotheses of bacteria having 'other' unidentified physiological functions; however, whether or not they stand true is yet to be ascertained and remains under question. APH(9)-la presents itself as an interesting enzyme for structural studies for its single-substrate profile and its potential to broaden our understanding of aminoglycoside modifying enzymes.

1.4 Aminoglycosides

The similar enzymatic profiles of APH(3')-lIla and APH(9)-la along with their drastically different substrate profiles, the former with multiple and the latter with only one, introduce the new questions of why and how. The structures of co­ crystallized forms would provide the rationale behind their dissimilarity. Answering these questions warrants a closer inspection of their substrates, aminoglycosides. Aminoglycosides are a class of compounds that are characterized by its distinctive chemical structure, possessing aminosugars linked via glycosidic linkages. They can be categorized into three groups: 1) 4, 5-disubstituted deoxystreptamine 2) 4, 6-disubstituted deoxystreptamines 3) other exceptions. Those that belong to the first two categories, which are often times referred to as the 'true' aminoglycosides, have a central 6-membered aminocyclitol ring that has two amine sugars linked via glycosidic bonds at the 4 and 5/6 positions. The atoms of the central ring, referred to as the deoxystreptamine ring, are numbered from 1 - 6 (Fig. 1a). Those of the two amino sugar substituents are numbered l' - 6' for the 4-substituted moiety, and

6 1" - 6" for the 5 or 6-substituted moiety. Aminoglycosides such as neomycin (4, 5 - substituted) and kanamycin (4, 6 - substituted) are probably sorne of the recognizable compounds in these two categories. The third class is separated on its own for they are not truly aminoglycosides. These include streptomycin and spectinomycin that possess a streptidine moiety and an actinamine substituent in place of the usual 2-deoxystreptamine, respectively (Figure 1).

6' NH 2 (;i!LHO0 o 2" OH HO l' HO'NH ro,iO~~6 HN~NH2 2 3 1

Kanamycin

OH

HO~OH H)-NH2 HO HN

~ H NH2 o O'HO~N--( HO~~ OH NH 1 0 HO

Spectinomycin Streptomycin

Figure 1: Chemical structures of aminoglycosides. Moieties in red indicate deoxystreptamine ring in kanamycin, streptidine ring in streptomycin, and actinamine ring in spectinomycin.

7 The reason behind the variable antibacterial potency amongst aminoglycosides was probed and established by Benveniste et al using structure-activity studies [43]. The hydroxyl groups were found to contribute little or no part in their potency; however, the number of amine groups, as weil as their positions, was identified as being the determining factor in conferring the antibiotic their varying strengths. Diamino moieties positioned at 2' and 6' positions of an antibiotic were found to be the most potent, followed by 6'-amino and 2'-amino groups alone. A complete lack of amine moiety on the molecule exhibited the least potency. These observations led to the synthesis of slightly modified aminoglycosides in hopes of introducing them as alternatives to those that have succumbed to resistant bacteria. These semi-synthetic molecules structurally resemble the natural aminoglycoside molecule but due to the slight modification at a specific functional position, they are not processed by the aminoglycoside modifying enzymes (Figure 2). Surprisingly, sorne have seen considerable success in clinical applications. For example, arbekacin, which is a derivative of kanamycin B, has been extensively used in Japan as an antibiotic against MRSA (methicillin-resistant Staphylococcus aureus) [44, 45]. The emergence of aminoglycoside modifying enzymes has not completely abolished the usefulness of conventional aminoglycosides as antibiotics. In fact, they are still widely used, and their practical applications are several-fold, providing relief for many an infection. Kanamycin is effective against a host of Gram-negative nosocomial infections. Streptomycin, the earliest discovered aminoglycoside, remains to be the most reliable and used antibiotic for Mycobacterium tuberculosis infections [46]. Not to be overlooked is spectinomycin. It is the main drug of choice for treating Nisseria gonorrhoeae infections, especially showing its usefulness where j3-lactamase inhibitors failed, since many in this species produce j3-lactamase.

8 Oibekacin Arbekacin

Figure 2: Structures of semi-synthetic aminoglycosides.

The pracess of aminoglycoside action involves two steps; entry into the bacterium, the rate-limiting point, and subsequent binding to ribosomal RNA, its target. The influence of aminoglycosides on bacteria is first dictated by the kinetics of drug entry. Bacterial uptake of these molecules entails three steps [15]. Aminoglycosides exist in polycationic states due to the presence of the amine groups that become pratonated at or near physiological pH. This positive charge mediates its entry into the bacteria. The pracess begins with the aminoglycosides attaching to the negatively charged phospholipids, lipopolysaccharides, and other membrane prateins in Gram-negative bacteria, and to phospholipids and teichoic acids in Gram-positive bacteria [47]. This initial binding step causes the displacement of magnesium and calcium ions that bridge the adjacent lipopolysaccharides [48]. Consequently, this event imposes some stress on the overall architecture of the cell membrane, resulting in increased membrane permeability [49]. What follows next is the energy­ dependent phase 1 (EDP-I) uptake. Here, a small amount of aminoglycosides gains entry into the bacterium whilst utilizing the transmembrane potential fram

9 the membrane-bound respiratory chains [50]. These membrane potentials are essential for drug influx, which explains why anaerobes are intrinsically resistant to aminoglycosides [51]. Moreover, facultative anaerobes are also resistant to low concentrations of aminoglycosides [52]. The small amount of aminoglycosides that now occupy the cytoplasm of the organism then diffuse in the cell matrix, and eventually comes in contact with and binds to its target, the ribosomal RNA. It binds tightly to the 168 ribosome of the 308 ribosomal subunit, which leads to the misreading of mRNA (incorrect tRNA binds to the A position on the ribosome) and yields misfolded, non-functional proteins. Notably, during EDP-I, some of these misfolded proteins interact with the membrane that further increases its permeability. This facilitates the onset of the final stage in the drug influx scheme; the energy-dependent phase Il (EDP-II) uptake. During this step, additional aminoglycosides invade the bacterium, saturating ail ribosomes. This last event results in further misfolded protein and accumulation of aminoglycosides. Ali these processes culminate to eventual cell death (bactericidal) or inhibited cell reproduction (bacteriostatic).

It is important to note that spectinomycin, the substrate of APH(9)-la, has a bacteriostatic effect. Instead of killing the bacteria, it inhibits further growth and reproduction of bacteria. Its mechanism of action is not unlike to that of the bactericidal aminoglycosides. 8pectinomycin binds to the minor groove of helix34 of the 168 ribosomal RNA [53]. During normal translation, peptidyl-tRNA is translocated from aminoacyl site (A site) to peptidyl site (P site), which entails the movement of various elements of the ribosome, including helix34. 8pectinomycin binding to this particular helix creates steric hinderance about the area of movement, which results in severely restricted conformational change. This, in effect, inhibits the translocation of peptidyl-tRNA from A to P site and protein synthesis seizes, leading to bacteriostatic state. Pharmacological significance of bactericidal and bacteriostatic drugs has been the subject of much debate. In fact, there is a general consensus that the distinction between the two has little relevance in clinical situations. For example, treatment of meningitis

10 with bacteriostatic drugs such as chloramphenicol was shown to be as effective as bactericidal agents, while showing the added advantage of reduced incidents of endotoxins [54]. Despite this information, spectinomycin has not seen as wide a use as other bactericidal agents such as kanamycin. Spectinomycin, however, continues to be an integral part of treatment for Neisseria gonorrhoeae infections [55].

1.5 Future of Drug Design

The evolutionary impact of humans on the biosphere is tremendous, so much so that its effect is felt in ail sectors of human life, from medicine to agriculture. Brought on by the emergence of massive industrialization that fosters an environment for rapid adaptation and evolution for many organisms, bacterial resistance to antibiotics is now prevalent, not only for aminoglycosides but for ail other antibacterial drugs on the market. There is a constant 'tug-of-war' fought between humans and bacteria. For every antibiotic that is introduced, bacteria quickly develop an arsenal of resistance, and it is no denying that the war seems to be in favour of the microbes, with the effect of antibiotics slowly but surely becoming diminished over time. Decreased sensitivities to currently used aminoglycosides have become commonplace, with surging numbers of aminoglycoside resistant strains of bacteria in clinical isolates being reported at an accelerated rate worldwide [37, 56-59]. The majority are accounted for by the presence of aminoglycoside modifying genes. Most recently, a bifunctional enzyme, AAC(6')-APH(2"), with a novel catalytic profile was isolated form a clinical isolate of MRSA in Japan [44]. This enzyme was found to deactivate arbekacin, a widely used MRSA drug in Japan, with great efficiency. This new function was aUributed to a single base mutation that changed one amine acid from serine to asparagine in the phosphorylation domain. Arbekacin, as mentioned earlier, is a relatively new semi-synthetic derivative of kanamycin B (Figure 2). This is a prime example that shows the rapid nature of bacteria's

11 survival adaptation, and it also iIIustrates the reasons behind the decline in aminoglycosides' effectiveness and usefulness.

Efforts to revive aminoglycosides from its weakening trend have seen IiUle progress due in part to the more optimistic focus on beta-Iactamase inhibitor development [60]. The latter drug is considered to be a more ideal candidate for clinical use than aminoglycosides for its milder nephrotoxicity and ototoxicity problems on patients. Accordingly, no research on new drugs against aminoglycoside resistance has gone to the extent or in depth as those seen for j3-lactamase inhibitors. Moreover, antibacterial drug discovery and development in general has been experiencing lukewarm interest from pharmaceutical and biotechnology companies in recent years. Antibacterial drug development saw its heydays fi ft Y years ago; however, much of the drug market have shifted their focus on chronic disease treatment [61]. It is not surprising that many companies have disengaged themselves from the antibacterial field since the average time for bacteria to become resistant to new drugs is only four years, reflecting little in capital gain. Divided interests coupled with pharmacoeconomic factors are contributing to the slow but steady decline in the effectiveness of aminoglycosides as drugs.

Despite its decreased prowess, aminoglycosides still remain as one of the most commonly used antibiotics today. Seeking novel drugs that act against aminoglycoside modifying enzymes is thus critical in maintaining the current bacterial treatment reliable. Novel inhibitor designs for aminolgycoside phosphotransferases is at a starting point. Kinetic and structural characterizations of the se enzymes have just begun to become available in recent years. Crystal structures of two aminoglycoside phosphotransferases, APH(3')-llla and APH(3')-lla, are now known [24,62]. Both have been noted to have similar protein folds to eukaryotic protein kinases (ePK's). More specifically, ail these enzymes share a 'kinase fold' centered around the N­ terminal lobe and the central core subdomain consisting of similar residue

12 sequences; HGDxxxxN (where x represents any amine acid) in APH's [62] and (H/Y)RDxxxxN in Ser/Thr and Tyr protein kinases [24]. The knowledge of such structural homologies centering on nucleotide binding regions amongst many kinases, suggesting a universal phosphoryl-transfer mechanism, has prompted the testing of known ePK inhibitors on APH(3')-llla [29]. Its kinetic characterization revealed that the enzyme is sensitive to eukaryotic protein kinase inhibitors, especially to isoquinolinesulfonamide groups that include the likes of CKI-7 and CKI-8 (Figure 3). These compounds act competitively with ATP to inhibit eukaryotic protein kinases [63]. Daigle et al showed that this class of inhibitors are also competitive inhibitors of APH(3')-lIla [29].

CI CI

CKI-7 CKI-8

Figure 3: Eukaryotic protein kinase inhibitors, CKI-7 and CKI-8.

The structural reasoning behind their inhibitory effect is now undergoing [64]. Comparison between ePK and APH classes of enzymes is important for identifying common as weil as distinctive features that can be exploited in future drug designs. Compounds that have differential impact on the various kinases within the host and the bacteria are critical part of successful drug targeting. In

13 fact, ATP binding in the respective enzymes has been noted to have marked differences [65]. APH(3')-lIla complexed with ADP revealed that the nucleotide is held in place via a pi-pi interaction between tyrosine-42 residue and the aromatic ring of the adenine. In contrast to this, nucleotide binding in cAPK involves a confined hydrophobic groove, where a tyrosine residue in its counterpart is replaced by an alanine residue [66]. Here, binding is believed to be largely due to fluctuating dipoles of the atoms. The rationale behind an ePK inhibitor such as CKI-7 inhibiting APH enzymes is unknown. The structural information would provide some clues to this common inhibitory effect, which would certainly be a starting point in selectively targeting APH class of enzymes.

1.6 Legionella pneumophila

The growing number of resistant bacterial strains continues to pose serious health hazards, as drugs designed to ameliorate their effects are persistently being challenged and defeated. The onset of widespread infections by Legionella pneumophila is a relatively new problem put forth into the medical community. Legionella pneumophila is a Gram-negative bacterium, and it is the major causative agent of Legionnaire's disease and Pontiac fever [67]. It inhabits natural and man-made aquatic environment both as a free-living organism and a protozoan parasite. Its parasitic nature extends to humans, as it can travel in aerosols and become consumed into the lungs of immunocompromised individuals. It is engulfed by alveolar macrophages that normally kill bacteria, but instead Legionella pneumophila continue to reside and multiply in number, and eventually lyses and spreads to other areas [68, 69]. Its potency is known to increase when it utilizes the amoeba as the conduit in between host infections [68]. Amoeba functions as the bacterial reservoir, where Legionella multiplies to a great number and lyses the host. Those that had been Iysed from amoeba were reported to be more efficient at subsequenthost entry (Le. humans). In addition, bacteria grown in these amoebic cysts exhibit highly resistant behaviour to biocides (such as chlorine) and heat treatment, methods normally used to treat

14 public water systems [70]. These aquatic parasites are now ubiquitous in our water systems where the environment is conducive to bacterial growth [71, 72]. It is no wonder that reports of nosocomial Legionella infections are increasing.

1.7 Objective

The discovery and kinetic characterization of a new phosphotransferase, APH(9)­ la, ignited our curiosity in the biology of aminoglycoside modifying enzymes for it displayed several unusual characteristics that set itself apart from the conventional species. In addition to its chromosomal origin, APH(9)-la is known to phosphorylate one aminoglycoside, spectinomycin, which is in stark contrast to the plasmid-borne, multi-aminoglycoside detoxifying APH's such as APH(3') subclass of enzymes. These findings prompted us to search for a structural reasoning behind its substrate specificity and a possible alternative physiological function that may explain their chromosomal origin. The crystal structure of the ternary complex allowed us to identify the key aminoglycoside binding residues. Most interestingly, the ternary and apo structures clearly displayed a difference in their respective lobe positions, hinting to a conformational change during catalysis. Our assays using substrates of homologous enzymes showed APH(9)-la is very specific for spectinomycin. Collectively, this study provides further insight to the enzyme's substrate specificity and the possible biological implication of the observed conformational change.

IS CHAPTER 2: Experimental Procedures

2.1 Materials

Ali reagents and chemicals were purchased from Sigma Aldrich Co. unless otherwise noted.

2.2 Protein Purification

Recombinant APH(9)-la containing a his-tag at its C-terminus was expressed in BB101(OE3) E. colistrain. 1-litre of Luria-Bertani broth containing ampicillin (100 IJg/ml) was inoculated with 12 ml of the overnight culture (BB101(OE3)/pET21b­ aph9) and grown at 37 oC with gentle shaking (240 rpm). At 00600 - 0.2, the temperature was reduced to 16 oC and the culture was allowed to incubate until

00600 - 0.5, at which point, the cells were induced with 1 mM IPTG. The cells were allowed to incubate a further 16 hours, after which, they were harvested by centrifugation (5000 x g). The cell pellet was resuspended in 50 ml of Buffer A (50 mM TRIS-HCI, pH 8.0, 300 mM NaCI, 10 mM Imidazole) supplemented with 1 mg/ml lysozyme. The cell suspension was incubated on ice with mild agitation for 30 minutes, and it was subsequently sonicated and centrifuged (10,000 x g) to obtain a clear cell extract. The Iysate was applied to Ni-NTA column that had been pre-equilibrated with Buffer A. The flow rate was maintained at 1 ml/min throughout the chromatography purification. The column was first washed with Buffer B (50 mM TRIS-HCI, pH 8.0, 300 mM NaCI, 250 mM Imidazole) at a low gradient (0 - 12 %). APH(9)-la was finally eluted with 100 % Buffer B. Purity was verified on a 12 % SOS-PAGE gel, as was evidenced by the presence of a single band at a molecular weight of 38 kOa. Pure fractions were pooled and concentrated using a Vivaspin centrifugai concentrator (MWCO 10,000). Buffer was exchanged to 25 mM HEPES, pH 7.5 by alternate concentrations and dilutions. This procedure gave a yield of approximately 20 mg of protein per litre of culture.

16 2.3 APH(9)-la Co-Crystallization with ADP and Spectinomycin

APH(9)-la (15 mg/ml) was co-crystallized in the presence of 5 molar excess of AOP and spectinomycin using hanging drop vapour diffusion method. Equal volumes of APH(9)-la/ AOP/ spectinomycin mixture and crystallization reagent (8% PEG8000, 0.2 M magnesium acetate, 10 mM manganese chloride, 0.1 M bicine, pH 8.0,) were combined on a siliconized glass cover-slip and hung over

700 ~I of the crystallization buffer at 4 oC. Crystals appeared within two days and grew to a size of 0.6 mm x 0.6 mm x 0.3 mm in 7 days.

2.4 Data Collection and Processing

APH(9)-la crystals were cryo-protected by first soaking it in 10 % ethylene glycol for 1 minute, then streaking it over a homogeneous mixture containing 25 % paraffin oil and 75 % Paratone-N (Hampton Research Corp.). The crystal was immediately flash-frozen in liquid nitrogen. The 2.5 A resolution data were collected from a single crystal at 100 K using X8C beamline equipped with AOSC Quantum-4R CCO detector at the NSLS (National Synchrotron Light Source) Brookhaven National Laboratories, NY. The diffraction data was processed using HKL2000 suite of programs [73]. The crystal belonged to P3(1)21 space group, with unit cell dimensions described in Table 1.

2.5 Structure Determination and Refinement

Phases were obtained by molecular replacement, using the apo APH(9)-la structure as the model. Phaser, a program that is part of the CCP4 package, was used to solve the phases [74]. Refinement was performed using CNS, with 9.5% of the data reserved as a cross-validation formula to monitor the progress of refinement (as Rfree). Initial refinement involved one round of simulated annealing, which yielded a starting Rfree of 38.3%. Substrates, AOP and

17 spectinomycin, were added to appropriate electron densities using parameter and topology files from PRODRG server [75]. Subsequent rigid body refinement further lowered the Rfree to 33.5%. Several cycles of individual B-factor refinement and energy minimization followed. 2Fo-Fc and Fo-Fc maps were calculated after each refinement step and inspected. Side chain positions were adjusted using a rotamer library to better fit with the electron density. Water molecules were added using the water-pick program in CNS, assuring that the molecules were within hydrogen-bond distances. The goodness of the structure was assessed by PROCHEK [76]. The structure was compared with that of the monomer B from the apo APH(9)-la structure to correct the backbone conformation that was initially in the disallowed region on the Ramachandran plot. Residue side chains with insufficient densities were assumed to be disordered and were changed to alanine or glycine. Further rounds of individual B-factor refinement and energy minimization lowered the Rfree to 29.8%. The high Rfree is due to the rather high B-factor values and to some crystal damage caused during the cryo-freezing step. Ali statistical values are summarized in Table 1 (Page 20).

2.6 Enzyme Inhibition Kinetics

APH(9)-la was purified as described in section 2.2, except in the final step of buffer exchange, 50 mM TRIS-HCI, pH 8.0 was used to be consistent with the APH(9)-la kinetics procedure of Thompson et al [19]. Steady-state kinetic study was performed by coupling the release of ADP from phosphorylation step by the kinase to pyruvate kinase and lactate dehydrogenase reactions as previously reported [21] (Appendix B). The assay buffer consisted of 50 mM TRIS, pH 7.5, 40 mM KCI, 10 mM MgCI2, 0.5 mg/ml NADH, 2.5 mM phosphoenolpyruvate, 3.5 units of pyruvate kinase, 5 units of lactate dehydrogenase, 1 mM spectinomycin, and varying concentrations of ATP. The reaction was carried out in a 96-well

plate, with a final reaction volume of 200 1-11. The assay buffer was pre-incubated for 5 minutes at 37 oC, and the reaction was initiated with the addition of 5 I-Ig of

18 1 1 purified APH(9)-la. The decrease in NADH (E = 6.22 mM- cm- ) absorbance was monitored at 340 nm using SPECTRAMax 190 (Molecular Devices) 96-well plate spectrophotometer. The range of ATP concentrations were kept between one­ fifth to five times the final Km. The concentration of spectinomycin was kept at near saturation level (1 mM). Inhibition experiment was performed at four different CKI-7 (dissolved in neat DMSO) concentrations. At each concentration, Michaelis-Menten parameter, Km apparent, was determined by best fits to non-linear least fit squares with its errors calculated using Grafit 3.0 [77]. The inhibition constant, KI, was determined fram a plot of Kmapparent versus CKI-7 concentrations, with the x-intercept giving the value of -KI (Appendix A). AI! assays were conducted in triplicates.

2.7 Substrate Assays

Enzyme assays were conducted on single-cel! UV-Vis Spectrophotometer (Varian) equipped with a thermo-equilibrated cel! block. Four different substrates were tested: choline, ethanolamine, glyceral, and methylthioribose (donation from Dr. Michael Riscoe, VA Medical Center, Portland, Oregon). Kanamycin and streptomycin were used as negative contrais. Ali assays were conducted in triplicates using the aforementioned coupled assay, with the final reaction volume of 1 ml. The concentration of ATP was maintained at 1 mM while those of the test substrates were kept at 0.1 mM.

19 Table 1: Data collection and refinement statistics

Data Collection APH(9), ADP, Spectinomycin Complex

Space group P3(1)21

Unit cell dimensions (A)

A 74.979

B 74.979

C 140.066

Resolution range (A) 50.0 - 2.5

Unique reflections 17,846 (1875)

Redundancy 9.3(6.1)

Completeness (%) 95.3 (87.5)

<1101> 12.98 (4.15)

a Rmerge 9.1 (37.0)

Refinement statistics

Number of atoms

Protein 2656

Substrate 50

Number of water molecules 95

Rcryst (%)b 24.4

Rfree (%)C 29.8

2 Average B-factor (A ) 52.4075

20 a Rmerge =rl(lhkl) - I/(rlhkl), where Ihkl is the integrated intensity of a given reflection. b Reryst =(rlFo - Fe)/(rFo), where Fo and Fe are observed and calculated structure factors. c 9.2% of reflections were excluded fram refinement to calculate Rfree. d Numbers in brackets denote values for the highest resolution bin

21 CHAPTER 3: Results

3.1 Protein Purification

The his-tagged (C-terminus tag) clone, BB101/pET21b-aph9, provided an excellent protein yield with a facile one-step purification procedure using Nickel­ NTA agarose resin. Low gradient wash with Buffer B removed ail non­ specifically bound proteins that included endogenous E. coli proteins as weil as protein degradation products (Figure 4a, peak 2). Elution with 100 % Buffer B gave a single sharp peak (Figure 4a, peak 1) from which the goodness of protein quality was confirmed by the presence of a single clean band at 38.5 kDa corresponding to APH(9)-la on 12 % SDS-PAGE gel (Figure 4b).

2 100%

%Buffer B

o Fractions

(a)

22 kQa M 1 2 1U3·tIi_, 66,4 .ij"ii' " ~

45:0 3.5.0

25;0

tEt4 1~.4

(b)

Figure 4: APH(9)-la Purification.

(a) Chromatogram of APH(9)-la purification using Ni-NTA resin. Blue line indicates absorbance at 280 nm, and the red line indicates % Butter B gradient. Peak 1 was pooled and used for crystallization and enzyme assays. (b) Coomasie-stained 12 % SDS-PAGE gel picture of purified APH(9)-la. Lane 1 indicates the pooled fractions from peak 1, and la ne 2 indicates protein from peak 2.

23 3.2 Co-Crystallization

Crystallization was performed using hanging-drop vapour diffusion method. Initial screening with commercially purchased crystallization buffers (Crystal Screen I/!!TM from Hampton Research Corp. and Wizard I/!!TM and Cryo 1111 ™ from Decode Genetics Inc.) yielded no crystals or promising leads; therefore, a systematic screening method was employed, wherein ail possible combinations of pH, buffers, salts, and PEG's (polyethylene glycols) at various protein concentrations were explored at 4 and 22 oC. A three-dimensional crystal appeared in a screening solution containing 15 % PEG8000, 0.1 M Bicine, pH 8.0 in 5 weeks. Further fine-screening involved narrowing down the optimal PEG percentage and introducing salts and additives (Additive Screens l, Il, III from Hampton Research Corp.). Presence of 0.2 M magnesium acetate along with 10 mM manganese chloride as an additive led to the improvement of the overall quality (Figure 5) of the crystal and hastened crystallization process (Iess than one week).

Finding the right freezing condition for the crystal entailed further crystallization optimization (Appendix C). 20 % glycerol, so often used as a cryo-protectant, did not fare weil with the APH(9)-la crystal. Exposure to solution containing the mother liquor with 20 % glycerol resulted in immediate degradation and eventual dissolution of the crystal. A plethora of cryo-protectants was tested, which included low-molecular weight polyethylene glycols, salts, and alcohols; however, in the presence of any one of the se compounds, the crystal showed rapid degradation. One compound, Paratone-N, was found to be the best solution for this problem. The rather high viscosity of Paratone-N was reduced by adding sorne paraffin oil, which facilitated the application of the crystal through the cryo­ protectant with little loss of crystal integrity.

24 Figure 5: Micrograph of APH(9)-la co-crystallized with spectinomycin and ADP.

3.3 Ternary Structure of APH(9)-la

Overall Architecture

APH(9)-la was crystallized in complex with ADP and spectinomycin, giving the overall crystal dimensions as described in Table 1. It belonged to space group P3(1 )21, with one molecule occupying the crystallographic asymmetric unit. The phases were solved by molecular replacement using the atomic coordinates of the apo APH(9)-la structure. The final model was refined to statistical Rfree and

Rcryst values of 29.8 % and 24.4 %, respectively, with zero residues in disallowed region as was assessed from Ramachandran plot using PROCHEK. Residues 328 - 331 were deleted from the structure due to insufficient electron densities.

25 Figure 6: Overall architecture of APH(9)-la in complex with ADP and spectinomycin. Spectinomycin and ADP are coloured green and dark blue, respectively. oC is coloured light blue. The N-terminus, C-terminus, helices, and strands are labelled accordingly.

*AII structure figures were prepared using PyMol [78].

26 Figure 7: Sectional representation of APH(9)-la. The N-terminal lobe is coloured orange. The C-terminal lobe consists of three regions: central core region (yellow), insert region (cyan), and the a-helix region (blue). Spectinomycin and ADP are coloured green and dark blue, respectively.

** For purpose of clarity and facile comparison, the notations assigned to subdivided sections of C-terminallobe of APH(9)-la are consistent with those used in the previously published structures of APH(3')-llla [24] and choline kinase [79].

27 The pratein has an overall U-shaped structure (Figure 6) composed of two identifiable lobes; a ~-strand-rich N-terminus (residues 3 - 101) and an o-helix­ rich C-terminus (residues 116 - 327). These two sections are connected by a 14-residue linker peptide (residues 102 - 115). The N-terminallobe begins with an unstructured peptide (residues 3 - 6) followed by a 310 helix-1 (residues 7 -

19), afterwhich a continuum of4 anti-parallel ~-strands (~1, ~2, ~3, ~4), encompassing residues 24 through 59, en sue that completes a ~-sheet motif.

The ~ strands exhibit a slight twist centered on ~2 and ~3. A short helix-2

(residues 60 - 73), which is inserted in between ~-strands 3 and 4, lies on the

opposite side of the ~-sheet with respect to helix-1. A long flexible loop connects

02 and ~4. ~4 is followed by a semi-beta structured peptide that runs acrass the width of the molecule. The overall structure of the N-terminus lobe is punctuated

by an obvious cleft formed by the ~-sheet that folds over the top of the cavity, the beta-structured linker residues (102 - 115) that outlines the side of the molecule,

and the two ~-strands fram the C-terminus lobe (~5 and ~6) running anti-parallel to each other (residues 211 - 223 and 224 - 232) that define the base. The somewhat larger C-terminal lobe consists of a series of short and long alpha­ helices with intermittent beta-strands, delineating the overall architecture of the protein. The C-terminal lobe can be divided into three sub-domains (Figure 7): 1) the core sub-domain consisting of four helices (03, 04, 05, and 06) and four

strands (~5, ~6, ~7, ~8) 2) the insert region comprised of helices A and B 3) the alpha-helix subdomain made of helices C and D. The core subdomain starts after the linker residues. Helix-3 is positioned at the base of the subdomain and

is followed by a short 1.5-turn 04. ~-strands 5/6, and 7/8 run anti-parallel to each other, with strands 5/8 positioned at the back of the molecule. Long peptides

connect ~ strands 5/6 and 7/8, positioning strands 6/7 in the vicinity of ~-sheet

motif of the N-terminus lobe, just beneath the linker peptide. The ~-strands are followed by two helices (05 and 06), with the latter Iying adjacent to 03. A striking feature of the C-terminallobe is the two sets of o-helices, each of which contains two helices that run anti-parallel to each other (residues 148 - 160, 170 -199,274 - 298, and 305- 319), comprising the insert and the o-helix regions.

28 The insert subdomain consists of helix-A and helix-B. aA is a small 2.5-turn helix. It is followed by aB, which is marked by a kink in the middle of the helix that projects outwards. The a-helix subdomain that follows from a6 of the central core region is comprised of two helices, aC and aD. Helix-C lies slanted towards the core subdomain in between helices A and D. It is followed by aD, which is interrupted by a 5-residue loop before the final helix turn. Helix-C is cradled in between helices A, B, and D, with hydrophobic interactions between the aromatic and branched side chains of the interacting helix faces providing much stability. These three helices seem to lend structural support to aC. Helix-B, which is divided into two sections by the bend in the middle of the helix, acts much like a structural brace that holds the long aC at the top and the bottom. In addition, helices A and D are positioned at the back and front of helix-C in a crossed fashion, providing further architectural support to the a-helix subdomain.

Aminoglycoside binding

The 2Fo-Fc electron density map shows clearly defined densities for both ADP and spectinomycin (Figure 8). The aminoglycoside, spectinomycin, is bound in the C-terminal region of the molecule via an extensive hydrogen bond network that extends from four residue side chains, four water molecules (Figure 9 and 11). The face of the fused three rings of spectinomycin lies parallel to the base of the enzyme, with the length of the molecule lined perpendicular with respect to the a-helices that run vertically beside it. Binding interactions come from the central core subdomain and the a-helix subdomain of the C-terminal lobe.

29 Figure 8: Observed electron densities of ADP and spectinomycin in a refined 2Fo-Fc map contoured at 1.00.

30 ...... , "

Figure 9: Spectinomycin binding site of APH(9)-la. Interactions between spectinomycin and enzyme residues are shown. Residues involved in binding are labelled and coloured with carbon atoms in orange, oxygen atoms in red, and nitrogen atoms in blue. Colouring in spectinomycin follows the same scheme except the carbon atoms are coloured white. Dotted blue lines indicate hydrogen bonds, while dotted red lines indicate interactions between magnesium ion and spectinomycin. Water molecules are depicted as blue spheres.

31 Spectinomycin can be divided into two sides, A and B, by cutting the molecule along the length of the molecule, to best demonstrate the various interactions that it makes (Figure 10). The 'A-side' is exposed to the a-helix subdomain, while the 'B-side' is exposed to the catalytic cavity.

B-side A-side

2.81 ,.$'OH ---2.-46----11 H20 1 ...... '

ASJ)212 (002) 2.46 Asp288 (001) 2 Mg + HO

~-.H o 0 2.90

HI""~! 4 .~"1I10H-----I.=.:....::=---I Asp288 (002)

o 1 : 4 OH L....:.:.":':::"-....j Arg284 (NH 1 ) ~C)-!

Figure 10: Schematic representation of hydrogen bond interaction between spectinomycin and APH(9)-la. The numbers indicate hydrogen bond distances in Angstroms.

32 Stabilizing interactions that hold the 'A-side' of spectinomycin come from a single helix, aC, with three of its residues (Arg284, Asp288, and Tyr292) participating in important hydrogen bond interactions (Figure 6,9, and10). One of the Arg284 amine arms (NH1) acts as the hydrogen bond donor to the hydroxyl oxygen attached to 4a carbon of spectinomycin. The carboxylate oxygen (001) of Asp288 acts as the H-bond acceptor to N-H hydrogen atom attached to C6, while its second carboxylate (002) accepts a hydrogen bond from the hydroxyl group attached at position 4a. The position of the two carboxylate oxygen atoms of Asp288 is further reinforced by hydrogen bonds to two nearby water molecules. The Tyr292 residue that is located just above Asp288 accepts a hydrogen bond from the C6 methylamine N-H hydrogen. It also acts as a hydrogen bond donor to the oxane oxygen at position 5. Asp212, which is part of the phosphotransferase motif, HxOxxxxN, dominates the interaction with the catalytically relevant 'B-side' of spectinomycin. The conserved motif resides on the loop that links 135 and 136 strands of the central core subdomain. The 9- hydroxyl group, which becomes phosphorylated by APH(9)-la, makes a hydrogen bond interaction with the carboxylate oxygen (002) of Asp212, as weil as with a magnesium ion. The cation also interacts with the phosphate tail oxygen of AOP and the 9-hydroxyl of spectinomycin. Interestingly, His210 and Asn217, which are also part of the conserved HxOxxxxN sequence, do not make any direct or indirect interaction with spectinomycin or AOP. Instead, both residues serve to lend stabilization to Asp212 (Figure 11). His210 hydrogen bonds to main chain amide of Asp212 and main chain carbonyl of lIe229, the latter of which is located on the loop connecting 137 and 138 strands that lie just adjacent to the HxOxxxN­ residing loop. The amine hydrogen of Asn217 interacts with the same carboxylate oxygen (002) of Asp212 that is hydrogen bonded to the 9-0H of spectinomycin. The position of Asp212 is further reinforced by hydrogen bonds between its carboxylate oxygen (001) and amine hydrogen (NH2) of Arg284 (Figure 11).

33 Figure 11: Interactions involving the conserved phosphotransferase motif, HxDxxxN. Dotted lines indicate hydrogen bond interactions between residues. Note that His210 does not interact with either of the ligands (spectinomycin or ADP).

34 Nucleotide binding

The ADP lies primarily in the N-terminus region, with its phosphate tail extending into the core region and meeting in close proximity to 9-hydroxyl group of spectinomycin at a distance of 4.77 A. The positions of a and f3 phosphates are stabilized by hydrogen bonds and salt bridges. The NZ of Lysine-52 makes a charge-charge interaction with the non-bridging oxygen atom of the a-phosphate, suggesting that this residue is involved in the positioning of the phosphate tail for enzyme catalysis. The f3-phosphate is stabilized by interactions with water molecules, Asn217, and magnesium ion. The amine hydrogen of Asn217 makes a hydrogen bond to the OD3 of f3-phosphate. Only one magnesium ion is present near the phosphate tail, with the prominent absence of the so-called primary and secondary divalent ions observed in APH(3')-llla and cAMP protein 2 kinase. The Mg + ion is positioned in a penta-coordinated fashion with its surrounding atoms, participating in several interactions (Figure 12). Most interestingly, the magnesium ion interacts simultaneously with the oxygen atom of the f3-phosphate of ADP and the 9-hydroxyl of spectinomycin, effectively acting as the mediator between these two ligands. The divalent ion is believed to play significant roles during catalysis [80]. Its coordination to five species is rare but not unprecedented for numerous crystal structures with penta-coordinated magnesium ions have been reported in the Protein Data Bank [81]. The aromatic rings of the nucleotide occupy a pocket lined with hydrophobic residues, lIe29, Phe50, lIe78, Phe102, lIe103, Leu219, and lIe229. The pl anar adenine rings face parallel to Phe50 located on f33 in a stacking fashion. This pi-pi interaction between the two provides a means for the proper positioning of the nucleotide within the hydrophobic cleft. Furthermore, a hydrogen bond interaction is observed between the N3 of the purine ring with the backbone amide of lIe1 03. Surprisingly, no hydrogen bond interactions exist for the ribose moiety of the nucleotide.

35 Figure 12: ADP binding. Magnesium and water molecules are depicted as red and green spheres, respectively. Residues involved in binding are coloured in orange. Carbon atoms in spectinomycin are coloured white. Carbon atoms in ADP are coloured light blue. Ali oxygen and nitrogen atoms are coloured red and blue, respectively. Dotted lines indicate interactions between the atoms of the residue and the ligands.

36 3.4 Inhibition Studies

To investigate the effect of eukaryotic protein kinase inhibitor, CKI-7, on APH(9)­ la, inhibition kinetics was performed using a coupled assay. Km apparent values at various inhibitor concentrations were determined, and a linear relationship was obtained between Kmapparent and CKI-7 concentrations (Figure 13a). Paralleling the APH(3')-lIla result, CKI-7 was found to inhibit APH(9)-la (KI = (159 ± 11) !lM) in a competitive fashion with respect to ATP (Figure 13b), although with 2.5 times less effectiveness. The experiment also confirmed the published Km value of APH(9)-la for ATP (Km = (19.9 ± 1.9) !lM) [19].

140

120 •

100

".... ~ 2: 80 -c: ~ a.CIl a. • CIl 60 E :::.::: 40

20

-200 o 200 400 600 800 1000 [CKI-7] (tJM)

(a)

37 26 24 .-... 22 ~ 20 J 18 Q) ~ 16 14 12 - 10 8 6 ~31~~~§:~~==::~====~~~ -0.040.02 0 0.020.040.060.08 0.1 0.120.140.16 1 1 [ATP] (IlM-1)

(b)

Figure 13: APH(9)-la Inhibition by CKI-7.

(a) Km apparent versus [CKI-7]. (b) Lineweaver-Burk plot. The assays were performed with the following CKI-7 concentrations: 0 (0),50 (e), 100 (0), 400

(.), and 800 (~) J.JM. Michaelis-Menten parameters were determined at each inhibitor concentration, and the Km apparent values were plotted against [CKI-7] to obtain the inhibition constant, KI.

38 3.5 Substrate Assays

Upon speculation of APH(9)-la's other possible substrates based on structural similarities to choline kinase (CK) [79] and methylthioribose kinase (MTRK) (private communications), four substrates were tested: choline, ethanolamine, glycerol, and methylthioribose. The tirst two are known to be substrates of CK, while the other two compounds are known substrates of MTRK [82, 83] Despite structural homologies to CK and MTRK, APH(9)-la did not show any phosphorylating activity on these four substrates (Figure 14). To verity the phosphorylating activity of APH(9)-la, assays using kanamycin and streptomycin were performed as negative contrais and spectinomycin as a positive control. Indeed, no phosphorylation was observed for the former two compounds, while high enzymatic activity with spectinomycin was detected.

6

4 ,-..

g 2 >.

:E 0.1 -<'-' '-' 0.08 ~ Q) r.% 0.06 0.04

0.02

0 ~~ .. ,t;>"" ~~ il' .. ~ .il' (Ji .§> ~ 0/"">

Substmte

Figure 14: Alternative Substrate Assay. Streptomycin and kanamycin were used as negative controls, and spectinomycin as a positive control. 1 unit (U) is the amount of enzyme that catalyzed the formation of 1 jJmole of product per minute at 37 oc.

39 CHAPTER 4: Discussion

We have determined the three-dimensional structure of aminoglycoside phosphotransferase(9)-la in complex with ADP and spectinomycin to a resolution of 2.5 A. In addition to the overall structurallayout of the enzyme, we now know the nature of its substrate binding. The structural information unraveled the mystery behind APH(9)-la's unusual single-substrate profile. More interestingly, comparison of the apo and ternary structures revealed a significant conformation al change, the tirst for this class of enzymes. Its structural and biological implication will be discussed below.

4.1 Comparison with APH(3')-lIIa

APH(3')-lIIa was the tirst aminoglycoside modifying enzyme to have its crystal structure solved. Comparison of APH(3')-llla and APH(9)-la shows that these two enzymes are different as much as they are similar. Despite having a low sequence identity of only 11 % (Figure 15), APH(3')-lIIa and APH(9)-la have similar structural layouts mostly centered on the N-terminal lobe and the central core region. APH(9)-la and APH(3)-lIla both have a \3-sheet motif sandwiched

between 310 helix-1 and a2, the latter ofwhich is linked to a linker residue that cradles the nucleotide. In addition, sequence alignment shows that both have an invariable lysine (Lys52 in APH(9)-la and Lys44 in APH(3')-lIIa) residue located on \33 that fold over the bound nucleotide (Figure 15). It is known that mutating this residue to alanine in both enzymes results in reduced binding affinity for ATP [19, 24]. Structural information clarifies this observation further. Lysine-52 in APH(9)-la lies just above the nucleotide, with NZ of this side chain making a salt­ bridge with the a-phosphate oxygen of the nucleotide (distance =3.42 A). Similarly, in APH(3')-lIla, the Lys44 that resides in \33 is located just above the nucleotide and makes a hydrogen bond interaction with the non-bridging a and \3 phosphates [23]. In conjunction with mutagenesis studies, this structural data

40 1 .....

",... APH(9j-Ia - -. ------KOPI OAOOLI ELLKVHYGI Dl H------TAOFI OGIlADT 33 APH(3')-lIla - - • - • - - - •• - •• - .-. - • - - • - A K MR l S PEL K K LIE KY· - - - • Re V K DT E •. MS 26 CKA.2 GMKELLSTMDLDTDANTIPELKERAHMLCARFLGGAWKTVPLEHLRISKIK.GMS g cAPK Q E SV/( E FLA KA K E lJ F L K K WE T P 5 Q N T A QI, D Q ------F D RI KT t GT.5 - F 43

110.. 110.. (X2 ...... ~~ APH(9)-Ia NA F A Y QA D S ------ES K S y FifrK Y ----- GY HD E 1 N L SI -- 1 R L L H 68 APH(3')-lIIa PA K V}' K L V G------EN EN L Y L· T D SR y K G - - - - - T l' Y D VER E K D MM 63 CKA-2 - NM.LFLCRLSEVYPPI RNEPNKVLL- - - - . Y- -- FNPE------TESBLVAESV 96 cAPK GRVMLVKHK------ESGNHYA LDKQKVVKL--KQIEHTLNEKRIL M ,1 ..... ",... ~ APH(9)-la DSGIKEIIFPIHTLEAKLPOQLKHFKIIAYPFIHAPNGFTQ------NLTGA0 il5 APH(3')-lIIa LWLEQKLPVPKV- - LHI/ERHD- - GWSNLLMSEADGVLCSEEYEDEO- -- -- SPEK 109 CKA-2 IFTLLSERH-LGPKLYGIFSG--GRLEEYIP---SRP------LSCHEISLAH D7 cAPK QAVNFP- FLVKL-- EFSFKON--SNLYMVMEYVAGGEMFSHLRRIGRFSEPHARF J~ ~ _ .(X3 - AftI...~~ ~ ~~--- APH(9)-la WKOLGKVLRQIH~TSVPISIQQQLRKEIYSPKWREI VRSPyNQIEFDNSD----- IS APH(3')-lIIa IIELYAECIRLFgSIDISD---CPYTNSLDSRLAELDYLLNNDLADVDC------Ig CKA-2 MS T KI A KRVAKVRQLE VP 1 WKEPDY L C EA L QRWLKOLTGTVDA E HR F DL P EECGV 192 cAPK y A A QI VI. TF E Y L .S L D ------150 ...... ~~4IIa.~ .___ r ..-,n'lff~~~~'--'" JB ~-.-.. ..-.x.-.~~ . ",... AI'H(9)-Ia --DKLTAAFKSFFNQNSAAiHRLVDTSEKLSKK1QPDLDKYVLC 215 APH(3')-lIla ------ENWEEDTPFKDPRELY- --- DFLKTEKPEEELVFS 194 CKA-2 SSVNC- -- LDLARELEFLRAHISLSKS------PVTF 229 cAPK ------L 1 160

......

",... APH(9)-la V Bv G N G E ------S 1 y 1 WD E ------231 APH(J')-lIIa 1 FVKDGK------VS GF G R ------210 CKA-2 1 rtiL P KAS S G N 1 R MS 1. S DE T 0 A ,. G N S L SA F NP A D P R 1. VI. E Y ------272 cAPK L ., D QQ G ------YI Q V T G F A KR V KG R T WL CG 188 lItro.. .a5 ...... ,.a6 --_ .." ~", -- APH(9)-la ------P ML A PliE RaL MF 1 GG G VG N V WN ------K P HE 1 0 Y F 261 APH(3')-IIla - - • ------S a RAD Mwy al A F C V R SIR E DI G ------• - - - E E 0 y V EL F 240 CKA-2 ------ASYNYRAFaFANHFJEWTIDyDIDEAPFYKIOTENFPENDOMLE 3M cAPE TPEYLAPEII LS KaYNfl.AvaWWALGVLI YEMAAGYPPFFADQPI QI YEKI VSaKV 243 aC ~_ aD ,..., ~ ~ .~~,...... ~ APH(9)-Ia y E G Y GAI N V D KT 1 L S y y R HE R 1 V E DIA V Y a 0 DL L S RN 0 A NA S R LES F K Y F K E MF D 316 APH(3')-lIla F D L L G - 1 K PD WE KI K Y YI L L DEL F ------• ------263 CKA-2 FFLNYLREOGNTRENELYKKSEDLVQETLPFVPVSHFFWGVWGLLQVELSPVGF G 3n cAPK RFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDI KNHKWFATTDWI AI YQRKV 298 l'ti .",."

..... APH(9)-Ja PNNVVEIAF-- 325 N APH(3')-llla 263 CKA-2 FADYGRDRLSLYFKHKOLLKNLA------.--0------­ 394 cAPK EAPFIPKFKGPGDTSNPDDYEEEEIRVINEKCGKEFTEF------o 337 Figure 15: Sequence alignment of APH(9)-la from Legionella pneumophila with APH(3')-llla from Enterococcus faecalis (POB 1J7L), Choline kinase-2A from Caenorhabditis elegans (CKA-2; POB 1 NW1), and cAMP-dependent kinase from Mus musculus (cAPK; POB 1BKX). Structure-based sequence alignment based on Levitt and Gerstein method was constructed using Indonesia software [84]. Secondary structure elements are shown as green arrows for l3-strands and red ribbons for helices. Residues shaded in dark grey and light grey are absolutely or partially conserved, respectively. Residues important for catalysis are boxed in red.

43 implicates Lys52 of APH(9)-la in correctly orienting the nucleotide phosphates for catalysis. Intriguingly, other interactions observed for the phosphate moiety in APH(9)-la present different pictures from those of APH(3')-llla and eukaryotic protein kinases [23, 66, 85]. In the latter two enzymes, two magnesium ions, the so-called primary and secondary cations, interact with the a and ~ phosphate anions that serve to balance the electrostatic charges between the negatively charged atoms of the enzyme residues and the nucleotide. In APH(9)-la, there is only one magnesium ion and a marked absence of the second cation (Figure 12). ln addition, the magnesium ion is penta-coordinated involving only one residue side chain (Asp230) in APH(9)-la, compared to the octahedral coordination involving two - residue interactions observed for APH(3')-lIla [23]. The sole magnesium ion occupies a space just in between the ~-phosphate oxygen and the 9-hydroxyl group of spectinomycin (site of phosphorylation), making a bridging interaction between these two species. In this respect, however, without the presence of the y -phosphate, the ADP/spectinomycin complex that we present here provides a limited sense of how ATP binding takes place. The stabilizing interactions between ATP and the enzyme, as weil as the orientation of the y-phosphate of ATP with respect to spectinomycin are not weil understood; therefore, the exact picture of the enzyme just before the phosphoryl transfer step is difficult to define. Fortunately, we have recently collected data on APH(9)-la in complex with AMPPNP, a non-hydrolyzable analogue of ATP, and its structure determination is currently under way (private communications). This structure will aid in providing a clearer view of the enzyme during catalysis.

Further to this discussion, nucleotide binding in APH(9)-la involves far less direct interactions with the enzyme residues, with only three residues, lIe1 03, Asn217, and Asp230, (Figure 12) compared to eight residues observed for APH(3')-lIla [23]. Of additional interest is the absence of a hydrogen bond between the 3'­ hydroxyl group of the ribose moiety to the residues of the linker region, which is present in APH(3')-llla [23]. Moreover, the ADP and magnesium ion have rather high thermal factors (as high as 89 A2 for ADP and 70 A2 for Mg2+) that points to

44 low stabilities of the ligand and ion in the binding pocket of APH(9)-la. These factors suggest that the nucleotide is poorly bound to the enzyme, which is likely due to the absence of the second divalent cation thought to be essential for catalysis and binding [80]. One might perhaps expect to relate this supposed 'poor binding' to its kinetic data; however, the Km(ATP) values reported for APH(3')-lIla and APH(9)-la are comparable, which provides no support for the observed poor binding in the latter [19, 21]. Kinetics information is thus not in line with the structural data, and so we suggest that the apparent poor binding of ADP is attributed more so to crystal packing than to its natural inclination for exhibiting such a binding manner. Despite the apparent poor binding, the nucleotide binding similarity between APH(9)-la and APH(3')-lIIa is still evident, and it is further reflected in the manner in which the adenine ring of the nucleotide binds. In addition to the lysine residues mentioned above, the ~3 strand of the N-terminal lobe contains a partially conserved aromatic residue (Phe50 in APH(9)-la and Tyr42 in APH(3')-llIa). The adenine rings of the nucleotide in both enzymes are buried within a cleft lined with hydrophobie residues. In APH(9)-la, the pl anar aromatic rings of Phe50 lies face to face with the rings of the adenine of ADP, with van der Waals interactions etfectively anchoring the nucleotide in place. This mode of nucleotide binding is reminiscent of the ring stacking observed for ADP and Tyr42 of APH(3')-llla [23]. What becomes obvious is that the overall protein folding patterns in the N-terminus of APH(3')-lIIa and APH(9)-la are similar, both having a defined hydrophobie cleft

with conserved residues on ~3 strand that provide paralleling interactions with the bound nucleotide. These features are perhaps reflective of their common enzyme mechanism, the Theorell-Chance mechanism. One other feature that ties APH(3')-lIla and APH(9)-la to this mechanism is the presence of the phosphotransferase motif, HxDxxxxN, in the core region. Central to catalysis is the invariant aspartate residue, which has been implicated to act as the general base that abstracts the hydrogen from the hydroxyl group of the aminoglycoside, thereby priming it for subsequent nucleophilic attack of the y-phosphate of ATP [18, 19]. Mutagenesis studies have weil established this residue to be significant,

45 for when mutated to an alanine, both APH(9)-la and APH(3')-lIla exhibited decreased kcat and increased Km values [19, 24]. The crystal structure of the former shows the carboxylate group of Asp212 making a hydrogen bond interaction with the 9-hydroxyl group of spectinomycin, the site of phosphorylation (Figure 9). This spatial arrangement and intimate association of the aspartate residue and spectinomycin clearly illustrates its importance for catalysis. Although the exact role of the aspartate residue in the phosphoryl transfer mechanism is unclear, the aminoglycoside-bound structure provided sorne evidence for the reason behind the mutagenesis results.

The roles of the two remaining conserved residues of the phosphotransferase motif, histidine and asparagine, are also now understood. The histidine residue was initially believed to take part in catalysis, possibly making a phospho-enzyme intermediate [86]. APH(3')-llla mutagenesis study of H188A, however, disproved this argument in which the mutant protein was reported to be extremely insoluble, and the mutation was found to have minimal effects on its activity [87]. This study showed that the histidine residue is not involved in phosphoryl transfer, but rather, its importance lies in providing intra-atomic interactions that are essential for proper protein folding [87]. Consistent with this observation, the crystal structure of APH(9)-la shows that the equivalent histidine residue, His210, does not make any direct interaction with either of the two ligands, spectinomycin or ADP. In fact, its interaction is confined to the main chain atoms of Asp212 and its neighbouring lIe229 residue, thereby lending structural integrity to the protein (Figure 11). The position of Asp 212 is stabilized via hydrogen bond to His210, which in turn is held in place by a hydrogen bond interaction to backbone carbonyl of lIe229. In addition, Asn217 hydrogen bonds to the carboxylate of Asp212. Thus, His210 and Asn217 act in concert to position Asp212 in line for catalysis, with the former providing anchoring force at the base, and the latter stabilizing the residue side chain at the top.

46 The major difference in the structures of the two APH enzymes centres o~ the C­ terminal aminoglycoside binding regions of the molecules. This is not at ail surprising given their different substrate profiles; APH(3')-llla being a multi­ substrate phosphotransferase while APH(9)-la being a single-substrate enzyme. To address their differences, it is important to recognize that APH(9)-la is a much larger molecule than APH(3')-lIla, having an excess of 67 residues. Sequence alignment of the two enzymes shows that the extra residues are reflected mainly in the C-terminus lobe, spanning the residues 303 - 331 (Figure 15). The extra 29 residues in APH(9)-la make up aC and aD. This helix-C is the distinguishing structural feature of APH(9)-la, for it is prominently larger than its equivalent in APH(3')-lIIa. Helix-D is completely absent in APH(3')-llla. The ternary structures of APH(3')-llla and APH(9)-la clearly show that there are two distinguishing features of aminoglycoside-enzyme interactions in their respective enzymes. First, the majority of the residues involved in aminoglycoside binding are not conserved in these enzymes. In APH(3')-lIla ternary structure, aminoglycoside­ binding force is dominated by acidic residues with 5 glutamate and 3 aspartate residues acting as hydrogen bond acceptors [25]. This predominantly negatively charged binding site is the marked feature in APH(3')-lIla. Furthermore, it has been noted that aminoglycoside binding in APH(3')-lIla does not make use of distinguishing functional groups of its various substrates for binding; as a result, no specifie interactions have been associated with aminoglycoside binding in APH(3)-lIla. It is these factors that have been suggested to confer APH(3')-lIla its ability to bind and detoxify a large number of aminoglycosides. By contrast, spectinomycin binding in APH(9)-la involves only two acidic residues, Asp212 and Asp288. In fact, we see that the residues lining the binding pocket in APH(3')-lIIa are not conserved at ail in APH(9)-la. Spectinomycin binding is dominated by hydrogen bond interactions from ordered water molecules and three residues (Arg284, Asp288, and Tyr292) from a single helix (aC). They take part in hydrogen bonds, spreading their interactions along the length of 'A-side' of spectinomycin (Figure 9). These specifie interactions highlight the role of aC in conferring stringent substrate selectivity of APH(9)-la. The lack of acidic

47 residues, in addition to the presence of the uniquely large oC, provides the structural basis behind APH(9)-la's unusual single-substrate profile. ln relation to the aforementioned difference between APH(3')-llla and APH(9)-la, their ternary structures also reveal that the residues involved in aminoglycoside binding in the two enzymes reside in different structural elements. In APH(3')­ ilia, they are spread in four different locations; the terminal turns of oC, 05, the loop structure that tethers aA and aB, and the central core region where Asp190 resides (Figure 16). By contrast, spectinomycin binding in APH(9)-la strictly involves only two structures; oC of the a-helix subdomain and the peptide that links 135 and 136 strands of the core region where Asp212 is located. That the interacting residues are so widely distributed within the C-terminal lobe in APH(3')-llla and concentrated on one helix for APH(9)-la is quite possibly a consequence of the different structures of each enzyme's phosphorylation substrate. For example, kanamycin, a substrate of APH(3')-lIla, is composed of three rings linked via glycosidic bonds, whereas spectinomycin, a substrate of APH(9)-la, is a relatively rigid molecule with three rings fused together (Figure 1). 'True' aminoglycosides, such as kanamycin, exist in different conformations [88], and thus perhaps their effective bindings require separate structural elements of APH(3')-llla to act in concert in order to accommodate the rather flexible nature of these molecules (Figure 16). For spectinomycin, since conformational change is limited due to its inherent rigidity, binding to the enzyme simply involves one helix (Figure 6).

4.2 Conformational Change

The apo and ternary structures of APH(9)-la reveal an apparent conformational change in the presence of substrates. There have been numerous structural studies that provide evidence supporting for the existence of 'open' and 'closed' conformations in eukaryotic protein kinases [85, 89]; however, the flexible nature of these enzymes is viewed with slight reservation owing to the argument of

48 crystal packing and laUice contacts that may necessarily put constraints on the protein conformation in exchange for overall low free energy of the crystal [85, 90]. As a result, whether or not the variable conformations are truly reflective of enzymes in catalysis in solution is left for interpretation. As for APH(9)-la, the crystals of apo and ternary forms were grown in completely different conditions, therefore, it is possible that the crystallizing reagents could have played a role in dictating their respective conformations. However, this argument becomes muted by the shear magnitude of the lobe movement, casting sorne doubt on the crystallization process for the observed 'closed' structure. Analysis by Difference Distance Matrix program (DDMP from Centre for Structural Biology at Yale University, New Haven, CT) showed that the domain hinge is situated approximately at residues 100 to 104, which corresponds to the linker peptide that connects the N-terminal and C-terminallobes (Figure 17). The lobe movement is undeniably substantial, with an estimated near 20° rotation that encompasses approximately 2 A. Superimposing the apo and ternary structures illustrates this phenomenon clearly (Figure 17). The N-terminallobe in the ternary structure is shifted more towards the central cavity than in its apo­ structure, effectively 'closing' the active site. DDMP analysis also indicated that the C-terminal lobe undergoes liUle conformation change in the presence of the two ligands, whereas the N-terminal lobe experiences sorne more localized changes. A closer inspection of the spectinomycin binding site shows that there are IiUle localized changes in the positions of the binding residues (Figure18). The distances between the ligand and the residues remain relatively unchanged. One notable difference is the Tyr292 ring flip. This shift effectively places the hydroxyl group of the tyrosine ring in favourable geometry and within hydrogen bond distance to the oxane oxygen (05) of spectinomycin (the distance change is from 3.55 A in apo form to 2.90 A in ternary form.) It also provides a good geometry for forming a hydrogen bond with the NH aUached to carbon-6 of spectinomycin. By contrast, there are more conformational differences in residues involved in ADP binding. Notably, the Phe50 makes an approximately 1 A shift downwards that places its aromatic ring in van der Waals interaction

49 distance with respect to the purine ring of the nucleotide (Figure 19). Another notable flexible region spans residues 28 - 38, which corresponds to the peptide that links f31 and f32. This is analogous to the loop region of APH(3')-lIIa and the GxGxxG motif in ePK that were reported to exhibit flexibility and display a shift away from the nucleotide binding site in the presence of a nucleotide [23]. In APH(9)-la, this loop folds over the top of nucleotide binding site, just as in APH(3')-llla (Figure 19). However, the loop shift observed in APH(9)-la is not as pronounced as one seen in APH(3')-llla (rms distance of 1.44 A for mainchain atoms of residues 28 - 38 in APH(9)-la compared to the reported 1.7 A for analogous loop region of APH(3')-lIla [23]). The loop structure in APH(3')-llla and ePK serves to provide stabilizing interactions to the phosphate group of the bound nucleotide either through hydrogen bonds between the residue backbone or the residue side chains. In APH(9)-la ternary complex, however, the residues in the polypeptide loop are not involved in direct interactions with the phosphates of ADP. The distances between the main chain atoms of the loop region and the phosphate tail are weil beyond hydrogen bond distance even after the shift in the ternary complex. Therefore, this loop's function in APH(9)-la is not for direct binding of the nucleotide but instead, it is in line with the suggestion posed by Burk et al. wherein the polypeptide was believed to provide a structural 'shield' for accommodating the hydrophobie binding cleft for the purine ring of the nucleotide.

The presence of substrates, ATP and spectinomycin, presumably induces a conformational change that brings ail the side chains closer together in the right conformation that allows stabilizing interactions to take place between the enzyme and the ligands. In addition to the binding interactions, the lobe movement brings the phosphate tail of the nucleotide and aminoglycoside phosphorylation site in close proximity, thus setting for subsequent phosphoryl transfer. The lack of conformational movement in APH(3')-lIIa was attributed to the close association of a2 and the loop between a4 and aA (Figure 16) via hydrogen bonds that in effect resulted in limited lobe flexibility [23]. This region

50 is, in fact, the domain hinge of the enzyme. In APH(9)-la, this interaction is not present, allowing for the flexible movement of the two lobes.

51 Figure 16: Ternary complex of APH(3')-lIla (PDS 1L8T). For simplicity, ADP and kanamycin A are not shown. Red indicates location of the do main hinge area whose structural elements are closely associated via H­ bonds. Green indicates the location of residues involved in aminoglycoside binding.

52 Figure 17: Ribbon representation of superimposed APH(9)-la structures. Apo and ternary structures are shown in grey and blue, respectively. The hinge region is indicated in red. The structures were superimposed with the C-terminal lobe in a fixed position using LSQMAN [91].

53 Figure 18: Magnified picture of spectinomycin binding site. Apoenzyme is shown in grey, and the ternary structure is coloured blue. Ali nitrogen and oxygen atoms are coloured blue and red, respectively. Spectinomycin is shown with its carbon atoms in white.

54 Figure 19: Magnified picture of superimposed APH(9)-la nucleotide binding site. Apoenzyme is shown in marine, while the ternary complex is coloured orange. [3- strands are numbered accordingly. ADP is shown with its carbon, nitrogen and oxygen atoms coloured purple, blue and red, respectively. N-terminallobes of apo and ternary enzyme were superimposed using LSQMAN [91].

The biological implication of lobe movement is poorly understood but it can be inferred by considering the roles of these enzymes in the organism. The roles and demands of various enzymes require different degrees of regulation, which may be reflected in the presence or absence of conformational changes.

55 APH(3')-llla is a ubiquitous enzyme, having been reported in numerous strains of bacteria. It is also a prolific enzyme, with the capacity to phosphorylate and thus detoxify up to ten aminoglycosides with good efficiency. Burk et al first noted the lack of conformational change in APH(3')-llla structures, and they suggested that this might be attributed to the enzyme being constitutively active [23, 25]. For times of sudden antibiotic influx, the enzyme being in an already 'closed' conformation allows it to accommodate rapid inactivation. The enzyme's highly demanding role in the organism may require that it be in a primed state and thus activity does not need to be tightly regulated, and hence its 'closed' state. By contrast, eukaryotic protein kinases (ePK) catalysis has been associated with significant domain movements [85, 92]. Eukaryotic protein kinases mediate the responses from external stimuli to phosphorylation of specifie targets, which requires tight enzyme regulation. It was suggested that the domain movement demonstrated by ePK's may be a secondary form of regulation at the protein level that confers an additional specificity, the primary being its own phosphorylation [23]. The lobe movement that we present here for APH(9)-la does not fit this reasoning; however, a sensible reason can be understood when comparing this to APH(3')-llla. We know that APH(9)-la's substrate identified thus far is spectinomycin, which is in stark contrast to the multi-substrate profile of APH(3')-llla. This drastically different phosphorylating activities suggest that APH(9)-la is not as rigorously required for the survival of the organism as APH(3')-lIIa is, and consequently it also does not need to be tightly regulated as in ePK's. Moreover, its lack of -10 and -35 polymerase recognition sequence upstream of aph9 gene along with the IiUle evidence to support for it being part of an operon cast doubts on whether or not the enzyme is at ail functionally present within the Gall. APH(9)-la, therefore, is not likely to be constitutively active like APH(3')-llla or under any tight regulation as seen in ePK. Instead, the apparent lobe movement is possibly a manifestation of its minimal role in the organism that would not require the enzyme to be in a primed position for catalysis at ail times. This would explain the 'open' form of the apoenzyme.

56 4.3 Comparison with Choline Kinase

When the crystal structure of choline kinase (CK) was solved, it was first noted that it resembled those of eukaryotic protein kinases (ePK) and APH's [79]. In addition, searches with DALI indicated that APH(9)-la is structurally most similar to choline kinase despite low sequence identity (6 %), yielding a Z-score of 10.9 and an overall rms deviation of 4.4 A[93]. Sequence analysis using Conserved Domain Database revealed that APH(9)-la has significant alignment (67.5 %) with choline kinase in residues 117 - 246 in APH(9)-la, which corresponds to the central core region of the protein [94]. What becomes apparent is that similarities between ail these three enzymes lie primarily in the N-terminal and central core subdomain nucleotide-binding regions (Figure 20). The N-terminallobe of CK presents a strikingly familiar protein fold composed of a five-stranded f3-sheet sandwiched between two helices [79]. Indeed, the f33 strand contains an arginine residue (Arg111) that is believed to be functi<;mally equivalent to the lysine residues of APH enzymes (Lys52 in APH(9)-la and Lys44 in APH(3')-lIla) (Figure 15) [95]. Along with the core subdomain, it defines a recognizable cleft where ATP would bind. The base ofthis cleft is formed by the core subdomain, just as in APH enzymes, which is lined with the conserved phosphotransferase motif (Y/HxDxxxxN) [35, 95]. They are the hallmark features of two-Iobed kinases, which hint to a common phosphoryl transfer mechanism. The salient aspartate residue that presumably forms a hydragen bond interaction with the nucleophilic hydroxyl group of substrate occupies the motif. By contrast, the C­ terminal lobe of CK bears little resemblance to APH(9)-la, and it is adorned with different lengths and arrangement of a-helices and loops than APH(9)-la (Figure 20). In addition, the C-terminallobe contains a faithfully conserved choline/ethanolamine kinase motif, [(LlV)X21D(FWY)E(YF)X 3NX3(FWY) DXsE)], located just downstream of the phosphotransferase motif that is absent in APH(9)-la [95]. Mutagenesis studies have demonstrated the importance of aspartate and glutamate residues in this motif for catalysis, and in particular, the raies of the aromatic residues such as tryptophan and tyrosine have been

57 suggested to play sorne role in choline binding via an electrostatic cation-TT interaction. With little similarities to spectinomycin binding residues, the choline kinase motif explains the substrate specificity displayed by APH(9)-la in the substrate assay conducted using substrates of its homologous enzymes (Figure 14).

c

(a)

Figure 20: Sectional view of (a) APH(9)-la and (b) Choline Kinase-2A (PDS 1NW1). Colour-coded to show N-terminus (orange), core subdomain (yellow), insert region (cyan), and a-helix subdomain (blue).

58 Unlike APH(9)-la, the apo structure of choline kinase exhibits a 'closed' conformation (Figure 20). Based on this observation and its structural similarities to APH(3')-lIla, Peisach et al. suggested that choline kinase activity most likely does not involve significant domain movement. In light of this information, an additional support for the requirement for conformational change begins to surface. The little or the lack of lobe movement in APH(3')-llla and as one suggested for choline kinase is likely a reflection of their highly demanding role within the organisms. Choline kinase is involved in the biosynthesis of phosphatidylcholine, a component of mammalian cell membrane, and it is necessarily present at a basallevel [82]. This is akin to APH(3')-lIla's suggested constitutive presence and activity in Enterococci bacteria discussed in the previous section. Thus, these enzymes probably evolved to have limited structural flexibilities that afford them the 'closed' conformation to maintain their catalysis-ready states. The opposite is expected for APH(9)-la whose 'open' apo conformation and lobe movement for catalysis are likely consequences of its low usage level in the day-to-day activity of Legionella bacteria.

4.4 Enzyme Inhibition by CKI-7 and Substrate Assays

Despite low sequence identities, the structures of APH(3')-llIa, ePK, and choline kinases clearly show that they are structurally homologous to one another and to APH(9)-la. Based solely on similar structural layouts and conserved residues, we asked the question of whether or not substrates of these enzymes are at ail shared amongst them. Similarity in structure translated to similarity in function only between APH(9)-la and APH(3')-llla.

CKI-7 is an isoquinolinesulfonamide group of ePK inhibitor. Earlier structural studies of APH(3')-llla led to an observation that this enzyme had a similar overall architecture to that of eukaryotic protein kinases [24]. High degree of similarity was noted in the N-terminallobe and the central core region, the latter of which contains the conserved phosphotransferase motif (Y/HxDxxxxN). This

59 observation led to the convincing finding that ePK inhibitors do inhibit APH(3')-llla [29]. Our assays revealed that CKI-7 also inhibits APH(9)-la activity in a competitive fashion with ATP, although with reduced potency than in APH(3')­ ilia. This reaffirms that CKI-7 binds in the ATP binding site, a region with the most semblance to APH(3')-llla and ePK's. Crystallographic study on CKI-7 binding to an ePK, casein kinase-1, demonstrated that the compound binds in the ATP binding pocket of its N-terminallobe [96], and it was further noted that the CKI-7 binding involves interactions analogous to ADP binding to APH(3')-lIla [29]. The nitrogen atom of the isoquinoline ring of CKI-7 makes a hydrogen bond to the hydrogen atom of the nearby main chain amide, much in the same way that ADP binds to APH(3')-lIla, with a hydrogen bond between the nitrogen atom of the purine ring and the nearby main chain amide hydrogen. Similarly, we now know from our structure that ADP binding in APH(9)-la also makes use of the same interaction (Figure 12). From this picture, we suggest that CKI-7 binds to APH(9)-la in a similar fashion as is observed for casein kinase-1.

Given the structural similarities between APH(9)-la and choline kinase-2A, in addition to the questionable biological function of APH(9)-la, we set out to explore the possibility of this enzyme sharing choline kinase's substrates. This is not the first such hypothesis to be tested, for APH(3')-llla was shown to phosphorylate substrates of serine protein kinases based on structural homology to ePKs and inhibitory effects of ePK inhibitors [29, 30]. Preliminary tests reveal that none of CK's substrates were compatible with APH(9)-la (Figure 14). This finding is not at ail surprising, for structural similarity between these two enzymes exist in the nuc\eotide binding area while obvious variability exists in the C­ terminal aminoglycosidel choline binding regions. Choline and ethanolamine (Figure 21) present different chemical structures, none of which resembles APH(9)'s substrate, spectinomycin. The latter contains a rigidly fused 3-ring­ structure that allows little, if any, substrate plasticity for binding to the active site of an enzyme. These different structures of the molecules would not allow the specifie hydrogen bond interactions from aC and HxDxxxxN of APH(9)-la to take

60 place. The similar protein folds in the nucleotide binding domains of the two enzymes suggest a common kinase origin, while the different C-terminal fold is a result of divergent evolution through which each enzyme underwent some changes to accommodate their respective substrates.

Nle /I~OH

Choline Ethanolamlne

Figure 21: Chemical structures of choline and ethanolamine.

61 CHAPTER 5: Conclusion

The crystal structure of APH(9)-la in complex with ADP and spectinomycin provided interesting structure-function relationships. The enzyme's tentative one-substrate specificity is conferred by a single helix (aC), which contains three unique residues involved in spectinomycin binding. The specifie binding is further explained by the presence of only two acidic residues in its binding, compared to eight acidic residues in APH(3')-llla. These two features are responsible for the substrate profile of APH(9)-la. In addition, the apo and ternary APH(9)-la structures displayed different conformations, hinting to an apparent lobe movement during catalysis, the first to be reported for aminoglycoside modifying enzymes. We propose that this conformational change is essential for catalysis that brings about the specifie binding of spectinomycin and nucleotide. APH(9)-la's structural homologies with other kinases such as eukaryotic protein kinases and methylthioribose kinases (private communications) had been noted previously, and of special note is the striking similarities with choline kinase. These observations posed the question of whether or not APH(9)-la had sorne other substrates other than spectinomycin that was physiologically important to the bacteria's survival. Assays indicate that the substrates of homologous enzymes are not phosphorylated by APH(9)-la. Its substrate specificity, chromosomal origin, and structural similarities to other kinases increase our curiosity, and the possibility of alternative substrates will continued to be explored. Furthermore, APH(9)-la was shown to be sensitive to eukaryotic protein kinase inhibitor, CKI-7. This information is in line with APH enzymes exhibiting structural similarity to ePK's centering about the nucleotide binding regions, namely the N-terminal and central core regions. This suggests that these two enzymes have a common ancestral origin. The ternary structure of APH(9)-la presented here provides interesting insights to its substrate specificity, and it also increases our understanding of APH class of enzymes that would aid in constructing a progressive avenue for designing novel drugs for therapeutic uses.

62 APPENDIX

A. Enzyme Kinetics

(equation 1)

Kapparent = K (1 + [1]) (equation 2) m m K 1

Data were fit to equation 1 with non-linear regression using Grafit software, and

Michaelis-Menten parameters, Km and kcat, were determined. The inhibition constant was determined by first obtaining the Km apparent values at different inhibitor concentrations. For competitive inhibition, a linear relationship exists between Kmapparent and [Inhibitor], in which the slope gives Km/KI, and the x­ intercept gives -KI. A plot of Kmapparent versus concentration of inhibitor was used to obtain KI.

63 B. Monitoring APH(9)-la reaction

ADP released from APH(9)-la phosphorylation was coupled to pyruvate kinase and lactate dehydrogenase reactions as shown. The decrease in absorbance of NADH was monitored at 340 nm to follow the overall reaction.

0- o 0- 0,/0- 0,/ ADP ATP NADH + H+ NAD+ ,/ 1 1 C-OPO -2 "--/ ~ H6-0H pyruvate kinase c=o "'-lactate dehydrogenaseL ~ 1 \1 3 1 CH2 CH3 CH3

phosphoenolpyruvate pyruvate lactate

Figure 22: Pyruvate kinase and lactate dehydrogenase-coupled reactions used for APH(9)-la assay.

64 C. Cryo-crystallography

Cryo-crystallography has come to replace room temperature data collection, for it offers advantages that are undoubtedly invaluable. Freezing the protein crystal and collecting its diffraction data at liquid nitrogen temperature allows the crystal to withstand the forces of x-ray radiation for a prolonged period of time. The life­ time of a crystal de pends on its buffering environment, pressure, and temperature. During x-ray exposure, the process of crystal decay is further hastened by the production and rapid transfers of free radicals. These radicals result from the photons' collisions with water molecules and atoms of the protein. Damages incurred by this phenomenon results in the disordering of the molecules within the crystal, which reduces the quality of diffraction spots, and hence a compromised data resolution. This problem can be reduced by cryo­ crystallography. Reaction rates are slowed down significantly at a temperature of 100K and consequently, the effects offree radicals are minimized during data collection. The advantages extend not only to betler diffraction qualities, but also to prolonged crystal life that allows data to be collected from a single crystal. This, perhaps, is the major obstacle that was overcome by cryo-crystallography since growing a crystal is the botlleneck in ail structure determination where initial growth and subsequent reproduction of the same crystal are almost always a challenge.

Just as with any biological samples, freezing a protein crystal requires the presence of a small molecule that can act as a cryo-protectant. Most often, crystallographers aim to grow crystals with a cryo-protectant already present in the mother liquor, which saves the problem of searching for the 'right match'. However, ail proteins are different and their compatibilities vary accordingly. Arriving at the best cryo-protectant can be accomplished by logic by increasing one of the precipitant concentrations; however, unpredictable crystal behaviours are frequently observed and as such, searching by extensive trial-and-error becomes inevitable. Cryo-protectants serve to replace the bulk solvent

65 surrounding the crystal and prevent the formation of ice upon freezing. Without a proper medium, diffraction due to ice can occur, which often results in and is associated with poor resolution.

66 LIST OF ABBREVIATIONS

Aminoglycoside acetyltransferase MC Aminoglycoside nucleotidyltransferase ANT Aminoglycoside phosphotransferase APH Adenosine Diphosphate ADP Adenosine Triphosphate ATP Area Detector Systems Corporation AOSC Charge Coupled Device CCO Collaborative Computing Project Number 4 CCP4 N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide CKI-7 1-(5-chloro-8-isoquinolinesulfonyl)-piperazine CKI-8 Crystallography and NMR System CNS Difference Distance Matrix Plot OOMP Dimethylsulfoxide DMSO 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEPES Isopropyl-thiogalactoside IPTG Lactate Dehydrogenase LDH 5' -Methylthioribose MTR Nicotinamide Adenine Dinucleotide (reduced) NADH Nickel-Nitrilotriacetic Acid NTA Sodium Chloride NaCI Polyethylene Glycol PEG Pyruvate Kinase PK

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74