STRUCTURAL STUDIES OF TERPENOID BIOSYNTHESIS AND BACTERIAL CELL DIVISION A Dissertation by DONG YANG Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2006 Major Subject: Biochemistry STRUCTURAL STUDIES OF TERPENOID BIOSYNTHESIS AND BACTERIAL CELL DIVISION A Dissertation by DONG YANG Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, James C. Sacchettini Committee Members, James C. Hu Patricia J. Liwang Terry L. Thomas Head of Department, Gregory D. Reinhart August 2006 Major Subject: Biochemistry iii ABSTRACT Structural Studies of Terpenoid Biosynthesis and Bacterial Cell Division. (August 2006) Dong Yang, B.S., Zhe Jiang University Chair of Advisory Committee: Dr. James C. Sacchettini The objective of this work is to investigate the structures of two nucleotide binding proteins: mevalonate kinase (MVK) and FtsZ. MVK is the key enzyme involved in terpenoid biosynthesis. In this study, we solved the crystal structures of the M. jannaschii MVK apoprotein, as well as the protein in complex with ligands. Its fold was analyzed and firmly established within the GHMP kinase family, in which homoserine kinase (HSK), phosphomevalonate kinase and galactokinase also belong. Structural analysis in combination with enzyme kinetics studies revealed the mechanism of this enzyme upon substrate binding, catalysis and inhibition. In particular, the phosphate-binding loop was found to be critically involved in the binding of nucleotides and terpenoids, via the interaction with a di-phosphate moiety from the ligand. An enzymatic reaction mechanism was constructed based on our structural data and it is consistent with kinetics studies from the literature. In this mechanism, the invariant residue Asp 155 functions as a general base that increases the nucleophilicity of the phosphoryl acceptor. Finally, a virtual screening study has been iv performed to explore the ligand binding potential of MVK. Compounds predicted to bind MVK were tested and analyzed. FtsZ is a prokaryotic homologue of tubulin that forms the apparatus for bacterial cell division. The structure of a crystal filament of the M. tuberculosis FtsZ complexed with GDP was described in this study. It shows an anti-parallel, left-handed double helical architecture. Compared with the straight crystal filament revealed earlier by other groups, the catalytic T7 loop in our structure is found to be outside the nucleotide binding site, indicating the GTPase is inactive. Furthermore, the buried surface area in our crystal filament is less, probably suggesting the helical FtsZ filament is less stable. We therefore proposed that the hydrolysis of GTP and the releasing of the γ-phosphate group will trigger the rearrangement of the FtsZ fibler, characterized by the exclusion of the T7 loop, which might lead to a less stable helical filament and would be the first step for the disassembly of FtsZ polymer. v DEDICATION To my parents and sister, for their love and support. To my friends for their help and encouragement. vi ACKNOWLEDGEMENTS First of all, I am thankful to Dr. James Sacchettini, my advisor, for giving me opportunities to do research in his lab and also giving me valuable advice and guidance during these years. I am also thankful to other members of my advisory committee: Dr. Patricia Liwang, Dr. James Hu and Dr. Terry Thomas, for their suggestions and discussion on my work. Second, I have to thank colleagues in Dr. Sacchettini’s lab, particularly Dr. Lance Shipman, as he led me into the world of crystallography when I just finished my rotation and joined this lab. I thank other members or former members in our group, particularly Dr. Remo Perozzo, Dr. Christoph Eicken, Dr. Sheng Ye, Dr. Clare Smith, Dr. Chih-chin Huang, Dr. Yoonsang Cho, Dr. Sudharsan Sridharan, Ms. Stephanie Swanson, Ms. Arati Ramesh and Mr. Feng Wang, for their advice and help at various stages during my work. My research also involves several collaborators. Particularly, I have to thank Dr. David Borhani (Abbott Bioresearch Center) and Ms. E. Lucile White (Southern Research Institute), for their initial work on FtsZ and their generosity in supplying me full-length FtsZ proteins. I have to thank Mr. Christos Savva for his kind help in performing EM studies on FtsZ filaments. I am also thankful to Drs. A. Ian Scott and Charles Roessner for providing us initial clones of MVK. Finally, my work could not be completed without help from the supporting staff in this department, particularly the administrative assistants for Dr. Sacchettni: Ms. Karen Hodges and the late Dr. Linda Fisher. vii NOMENCLATURE ADP Adenosine 5’-diphosphate ADPβS Adenosine 5’-(βthiol)-diphosphate AMP Adenosine 5’-monophosphate AMPPCP Adenosine 5’-(β, γ-methylene)-diphosphate APS Advanced Photon Source ATP Adenosine 5’-triphosphate CAMD Center for Advanced Microstructures and Devices CDP Cytidine 5’-diphosphate CDP-ME 4-diphosphocytidyl-2-C-methyl-erythritol CDP-ME2P 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate CMP Cytidine 5’-monophosphate CoA Coenzyme A CTP Cytidine 5’-triphosphate DEAE Diethylaminoethyl DMAPP Dimethylallylpyrophosphate DMSO Dimethyl Sulfoxide DTT Dithiothreitol dUTP 2'-deoxyuridine 5'-triphosphate DXP 1-Deoxy-D-xylulose 5-phosphate EM Electron Microscopy FPP Farnesyl Pyrophosphate viii GAP GTPase Activating Protein GDP Guanosine 5’-diphosphate GDPβS Guanosine 5’-(βthiol)-diphosphate GGPP Geranylgeranyl Diphosphate GMPPCP Guanosine 5'-(β, γ-methylene)-diphosphonate GMPPNP Guanosine 5’-(β, γ-imido)-triphosphate GPP Geranyl Pyrophosphate GTP Guanosine 5’-triphosphate GTPγS Guanosine 5'-(3-O-thio)triphosphate HEPES N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic Acid HIDS Hyperimmunoglobulinemia D Syndrome HMB-PP (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate HMG 3-Hydroxy-3-methyl glutaryl HSK Homoserine Kinase IPP Isopentenyl Pyrophosphate IPTG Isopropyl-beta-D-thiogalactopyranoside ITP Inosine 5’-triphosphate LB Luria Bertani Broth LDH Lactate Dehydrogenase LDL Low Density Lipoprotein MAD Multiwavelength Anomalous Dispersion MECDP 2-C-methyl-D-erythritol 2,4-cyclodiphosphate ix MEP Methylerythritol Phosphate MIR Multiple Isomorphous Replacement MPD 2-methyl-2,4-pentanediol MVK Mevalonate Kinase NAD+ Nicotinamide Adenine Dinucleotide (oxidized form) NADH Nicotinamide Adenine Dinucleotide (reduced form) NADPH Nicotinamide Adenine Dinucleotide Phosphate (reduced form) NCS Non-crystallography symmetry NDP Any Diphosphonucleotide PAGE Polyacrylamide Gel Electrophoresis PCR Polymerase Chain Reaction PIP Di-iodobis-ethylenediamine-diplatinum II nitrate PK Pyruvate Kinase PMSF Phenylmethylsulfonyl Fluoride RGS Regulators of G Protein Signaling SAD Singlewavelength Anomalous Dispersion SIR Single Isomorphous Replacement SDS Sodium dodecyl sulphate Se-Met Selenium Methionine SRE Sterol Regulatory Element Tris Tris(hydroxymethyl) Aminomethene UPPS Undecaprenyl Pyrophosphate Synthetase x UTP Uridine 5’-triphosphate X Any Amino Acid Residue xi TABLE OF CONTENTS Page ABSTRACT ……………………………………………………………………………iii DEDICATION ………………………………………………………………………….v ACKNOWLEDGEMENTS……………………………….……………………………vi NOMENCLATURE……………………………………………………………………vii TABLE OF CONTENTS……………………………………………………………….xi LIST OF FIGURES……………………………………………………………………xiii LIST OF TABLES……………………………………………………………………..xvi CHAPTER I INTRODUCTION TO STRUCTURAL BIOLOGY …………………....….1 Scope of Structural Biology…………………….…………………….....1 Introduction to Crystallography ...………..……………..……..………..5 Theory of Structural Based Drug Design ...…….………...…………...20 Overview of Chapters ……….………...………….……...……………24 II BACKGROUND OF TERPENOID BIOSYNTHESIS AND MVK……....27 Terpenoid Biosynthesis ……..……..…………………………….…....27 MVK…………………………………………..……......………….…..41 Significance of This Work………………………...…………………...47 III STRUCTURAL STUDY OF MVK………………………………………48 Methods ……………..….…...………………………………………...48 Results and Discussion……………………...…………………….…...56 Conclusion……………………………………………………………113 xii CHAPTER Page IV BACKGROUND OF CELL DIVISION AND FTSZ…………....……...116 Bacterial Cell Division ..……..……………………………….….….116 FtsZ ……..………………..……………………………………….....117 Significance of This Work…………………………………………...126 V STRUCTURAL STUDY OF A FTSZ CRYSTAL PROTOFILAMENT.128 Methods ……………………………………………..………………128 Results and Discussion ……………………………………….……..133 Conclusion …………………………………………………………..157 VI GENERAL CONCLUSION AND FUTURE DIRECTIONS …………158 REFERENCES………………………………………..….…..………………………..163 VITA…………………………………………………………………………………...175 xiii LIST OF FIGURES FIGURE Page 1. Basics of Diffraction.……….………………………………………………..……..8 2. Principle of MAD………………………….………..………………………….....12 3. Principle of Molecular Replacement……………..……………………………….14 4. The Mevalonate Dependent Pathway for IPP Biosynthesis….………………...…33 5. The Non-mevalonate Pathway for IPP Biosynthesis…………..……..…………..36 6. Prenyltransferase………………………………………………………………….38 7. The Reaction Mechanism of MVK……………………………………………….42 8. Multiple Sequence Alignment of MVKs from Various Sources…………….……58 9. Anomalous Patterson Map………………………………………………….…….62 10. The Electron Density Maps before and after Density Modification……....….…64 11. Rat and M. jannaschii MVK…………………………………………….………66 12. Ramachandran Plot of MVK Crystal Structure…………………..…….……….68 13. Fold Comparison of N-domains of GHMP Kinases……………….……………70 14. Fold Comparison of C-domains of GHMP Kinases……………….……………71