Structural studies on DdCAD 1 a ca2+ dependent cell cell adhesion protein

STRUCTURAL STUDIES ON DDCAD-1: A CA2+-DEPENDENT CELL-CELL ADHESION PROTEIN LIN ZHI (M.Sc.) A THESIS SUBMITTED FOR DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements I would like to express my sincere appreciation to my enthusiastic supervisor, Associate Professor Yang Daiwen, for his guidance, inspiration, patience and trust throughout this project Special thanks to Prof Siu Chi-Huang as well as my collaborators, Sriskanthadevan Shrivani and Huang Haibo, from Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto Without their efficient collaboration it would not have been possible for me to complete this project in time I would also like to express my appreciation to Dr Song Jianxing, Dr Mok YuKeung and other QE committee members, for their helpful advice and critical suggestions Thank Dr Mok for his kindly providing an over-expression vector used in this work Thanks were also due to Dr Fang Jingsong for his assistance in NMR experiments I wish to take this opportunity to express my gratitude to my fellow graduates, postdoctoral fellows, friends, brothers and sisters from department of biological sciences and other departments/institutes Their friendship made my research life at the NUS a pleasant learning experience In particular, I’d like to thank B C Karthik, I Chan Siew Leong, Dr Du Ning, Dr, Huang Weidong, Jiang Naxin, Dr K P Manoharan, Liu Yang, Li Kai, Dr Pung Yuh fen, Qiu Wenjie, Ran Xiaoyuan, Dr Ru mingbo, Siu Xiaogang, Wang Wun Long, Xu Xingfu, Xu Ying, Dr Xu Yingqi, Yang Shuai, Dr Zhang Jingfeng, Dr Zhang Xu, Dr Zhang Yonghong, Zhang Yuning, and Zheng Yu for their kind help in experiments and/or data analysis Although any words are not even enough to express my heartfelt gratitude to my family in China, I would still like to thank my parents for their sustaining family love and support Praise the Lord for His amazing grace and unfailing love Without this everlasting love, I would not have been able to accomplish or even start this thesis Lastly, the financial assistance in the form of a research scholarship provided by NUS is gratefully acknowledged II Table of Contents ACKNOWLEDGEMENTS I TABLE OF CONTENTS III LIST OF TABLES XI LIST OF ABBREVIATIONS SUMMARY XII XIV CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW 2.1 STUDIES ON DDCAD-1 OF DICTYOSTELIUM DISCOIDEUM: A CA2+- Biological background of D discoideum The genome of D discoideum The striking feature of D discoideum development Characterization of DdCAD-1 10 Molecular Characterization of DdCAD-1 10 Functional characterization of DdCAD-1 13 Adhesion properties of DdCAD-1 13 Expression of DdCAD-1 14 A Novel Transport Pathway 15 Involvement of DdCAD-1 in the regulation of developmental events 17 2.1.3 Summary 18 2+ 2.2 STRUCTURAL STUDIES ON PROTEIN S OF MYXOCOCCUS XANTHUS, A CA DEPENDENT SELF-ASSEMBLY MOLECULE FROM PROKARYOTE 19 2.2.1 Biological context 19 2.2.2 Structures of protein S 21 2.2.2.1 NMR structural studies on protein S 21 2+ 2.2.2.2 X-ray structure of Ca -bound NPS 24 2.2.3 Models for protein S multimerization 25 2+ 2.3 STRUCTURAL STUDIES ON CADHERINS: THE CA -DEPENDEND CAMS FROM EUKARYOTES 25 DEPENDENT CELL ADHESION PROTEIN FROM EUKARYOTE 2.1.1 2.1.1.1 2.1.1.2 2.1.2 2.1.2.1 2.1.2.2 2.1.2.2.1 2.1.2.2.2 2.1.2.2.3 2.1.2.2.4 III 2.3.1 The cadherin superfamily 2.3.1.1 Introduction 2.3.1.2 Classical cadherins 2.3.1.2.1 Type I cadherins 2.3.1.2.2 Type II cadherin 2.3.1.3 Desmosomal cadherins 2.3.1.4 Protocadherins 2.3.1.5 Cadherins-like subfamily 2.3.2 Structure-based models of cadherin-mediated cell adhesion 2.3.2.1 Model A 2.3.2.2 Model B 2.3.2.3 Model C 2.3.2.4 Model D 2.3.2.5 Model E 2.3.3 Summary 2.4 PROTEIN STRUCTURAL DETERMINATION BY NMR 2.4.1 Introduction to NMR phenomenon 2.4.1.1 Discovery of NMR 2.4.1.2 Basic principles of NMR 2.4.1.3 Basic parameters of NMR 2.4.1.3.1 Chemical shift 2.4.1.3.2 J-coupling 2.4.1.3.3 NOE 2.4.1.3.4 Chemical Exchange 2.4.1.3.5 Relaxation 2.4.2 Advantages of structural studies by NMR 2.4.3 The general strategy to NMR structure determination 2.4.3.1 Sample preparation 2.4.3.2 Recording NMR spectra 2.4.3.3 Resonance assignment 2.4.3.4 Collection of NMR restraints 2.4.3.5 Structure calculation and refinement 2.4.3.6 Evaluation of structural quality 2.4.4 Summary CHAPTER 3: MATERIALS AND METHODS 3.1 MEDIA 3.1.1 M9 medium 3.2 GENERAL DNA MANIPULATION 3.2.1 Agarose gel eletrophoresis 3.2.2 Preparation of plasmid DNA from E coli 3.2.3 DNA digestion, DNA fragment purification and ligation 25 25 28 29 29 30 31 32 33 33 36 37 39 40 41 42 42 42 43 44 44 45 45 45 46 47 48 48 50 50 51 51 52 53 54 55 55 56 56 56 56 IV 3.2.4 DNA sequencing 3.2.5 Polymerase chain reaction (PCR) 3.2.6 Construction of mutants 3.3 BACTERIA TRANSFORMATION 3.3.1 Preparation of E coli competent cells 3.3.2 Transformation of E coli competent cells 3.4 GENERAL PROTEIN MANIPULATION 3.4.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 3.4.2 Native PAGE 3.4.3 Quantitative protein assay 3.4.4 Protein secondary structure evaluation by Circular Dichroism (CD) 3.5 EXPRESSION AND PURIFICATION OF DDCAD-1 3.5.1 Expression vector 3.5.2 Expression of DdCAD-1 in E coli 3.5.2.1 Expression of unlabeled DdCAD-1 3.5.2.2 Stable-isotopic labeling of DdCAD-1 3.5.2.2.1 15N or 15N, 13C labeling 3.5.2.2.2 2H , 15N, 13C labeling 3.5.3 Purification of DdCAD-1 3.6 DETECTION OF DDCAD-1 HOMOASSOCIATION BY DLS 3.7 NMR EXPERIMENTS 3.7.1 One dimensional NMR 3.7.2 Two dimensional NMR 3.7.2.1 2D 1H-15N HSQC spectrum 3.7.2.3 2D 1H-13C HSQC spectrum 3.7.2.4 H/D exchange measurements 3.7.2.5 Ca2+/Mn2+-titration 3.7.3 Three dimensional NMR 3.7.3.1 HNCACB and CBCA(CO)NH 3.7.3.2 HNCO and HN(CA)CO 3.7.3.3 CC(CO)NH and H(CCO)NH 3.7.3.4 HCCH-TOCSY 3.7.3.5 Non-constant time 3D 15N-edited and 13C-edited NOESY 3.7.4 Four dimensional NMR 3.7.4.1 4D 13C,15N-edited NOESY 3.7.4.2 4D 13C,13C-edited NOESY 3.8 RESONANCES ASSIGNMENT 3.8.1 Backbone assignment 3.8.2 Aliphatic sidechain assignment 3.8.3 Aromatic sidechain assignment 3.9 NOE ASSIGNMENT, STRUCTURAL CALCULATION AND REFINEMENT 3.10 STRUCTURE-BASED ALIGNMENTS AND STRUCTURAL COMPARISON 3.11 MODEL STRUCTURE CALCULATIONS 57 58 58 59 60 60 61 61 61 62 62 64 64 64 64 65 65 65 66 68 68 69 69 69 70 71 71 72 72 72 73 74 74 76 76 76 76 76 77 77 78 79 79 V CHAPTER 4: RESULTS 81 4.1 EXPRESSION AND PURIFICATION OF DDCAD-1 82 4.1.1 Expression of His-DdCAD-1 in E coli 82 4.1.2 Determination of solubility of over-expressed His-DdCAD-1 82 4.1.3 Purification of DdCAD-1 82 4.1.3.1 The first Ni-NTA affinity chromatography 82 4.1.3.2 Thrombin cleavage 85 4.1.3.3 The second Ni-NTA affinity chromatography 85 4.1.3.4 Gel filtration 85 4.1.4 The final construct of DdCAD-1 89 4.2 IN-VITRO CA2+-BINDING PROPERTY OF DDCAD-1 89 4.3 THERMAL MELTING PROPERTIES OF DDCAD-1 93 4.4 NMR ASSIGNMENT OF DDCAD-1 98 4.4.1 Backbone assignment 98 4.4.1.1 Backbone assignment of Ca2+-free DdCAD-1 98 4.4.1.2 Backbone assignment of Ca2+-bound DdCAD-1 98 4.4.2 Aliphatic sidechain assignment 102 4.4.3 Aromatic sidechain assignment 102 4.4.4 Secondary structure characterization from backbone assignment 109 4.4.5 NOE assignment 112 4.5 NMR STRUCTURE OF DDCAD-1 117 2+ 4.5.1 NMR Structure of Ca -free DdCAD-1 117 4.5.2 NMR Structure of Ca2+-bound DdCAD-1 118 4.5.3 Proline conformation 123 4.5.4 Ca2+-binding sites 123 4.6 STRUCTURAL COMPARISON TO OTHER CELL ADHESION PROTEINS 126 2+ 4.7 CA -DEPENDENT HOMO-ASSOCIATION OF DDCAD-1 IN VITRO 136 4.8 SUBDOMAIN STUDIES 138 4.9 MUTAGENESIS STUDIES 143 4.10 STRUCTURAL MODEL OF THE CA2+-BOUND DDCAD-1 DIMER 147 CHAPTER 5: DISCUSSION & CONCLUSIONS 5.1 5.2 5.3 5.4 HEAT-INDUCED AGGREGATION OF DDCAD-1 AROMATIC SIDECHAIN ASSIGNMENT MECHANISM OF CA2+-DEPENDENT ADHESION MEDIATED BY DDCAD-1 DISTINCT ROLES FOR THE TWO DOMAINS OF DDCAD-1 CHAPTER 6: REFERENCES 152 153 153 155 156 160 VI APPENDIX I: STATISTICS ON PROTON-PROTON DISTANCES 191 APPENDIX II: REPRESENTATIVE SLICES FROM 13C & 15N-EDITED NOESY OF MBP 193 APPENDIX III: CONSTANT-TIME 13C-1H HSQC OF 13C, 15N-LABELED MBP 194 APPENDIX IV: SUMMARY OF ASSIGNMENTS OF AROMATIC RESONANCES OF SAM, DDCAD-1 AND MBP 195 APPENDIX V: DIFFERENT FUNCTIONS OF THE TWO DOMAINS OF DDCAD-1 196 APPENDIX VI: BINDING OF 45CA2+ TO WILDTYPE AND MUTANT HIS6DDCAD-1 PROTEINS 198 APPENDIX VII: LIST OF PUBLICATIONS RELATED TO THIS STUDY 199 APPENDIX VIII: LIST OF OTHER PUBLICATIONS 200 VII List of Figures Figure 2.1 The development of D discoideum and the expression of CAMs Figure 2.2 Sequence of DdCAD-1 11 Figure 2.3 Structures of Protein S 22 Figure 2.4 Schematic overview of the cadherin superfamily depicting representative molecules for the respective subfamilies 27 Figure 2.5 34 Structure-based cadherin models Figure 2.6 The general flowchart for determining solution structures of proteins by NMR 49 Figure 3.1 A pET-32a-derived expression vector 63 Figure 3.2 Pulse sequence for recording simultaneously 13C- and 15Nedited NOESY 75 Figure 4.1 Subcloning and over-expression of His-DdCAD-1 83 Figure 4.2 Purification of His-DdCAD-1 by Ni-NTA column 84 Figure 4.3 Thrombin digestion of His-DdCAD-1 at room temperature 86 Figure 4.4 Gel filtration profile of DdCAD-1 in the absence of Ca2+ (red) and in the presence of Ca2+ (blue) with high absorbance at 280 nm 87 Figure 4.5 88 SDS PAGE of final purified DdCAD-1 Figure 4.6 The final construct of DdCAD-1 sample 90 Figure 4.7 91 Q-TOF mass spectrum of DdCAD-1 Figure 4.8 In-vitro Ca2+-binding properties of DdCAD-1 92 Figure 4.9 Thermal denaturation curves of Ca2+-free/Ca2+-bound DdCAD1 as measured with CD at 212 nm 94 Figure 4.10 The effect of temperature on the conformation of Ca2+free/Ca2+-bound DdCAD-1 as measured with CD 95 VIII Figure 4.11 CD spectra of Ca2+-free DdCAD-1 after temperature denaturation 96 Figure 4.12 Apparent hydrodynamic molecular weight (MW) of Ca2+-free DdCAD-1 aggregate from DLS measurement at 20 ºC 97 Figure 4.13 C , C connectivity for a stretch of residues from P93 to F96 99 Figure 4.14 1H-15N HSQC spectrum of DdCAD-1 acquired at 500 MHz and 30 C on a sample of 0.8 mM protein and pH 6.2 100 Figure 4.15 1H-15N HSQC spectra of DdCAD-1 at 30 ºC and pH 6.2 in the absence (blue) and presence (red) of Ca2+ 101 Figure 4.16 Representative slices of aliphatic sidechain assignments of Ca2+-free DdCAD-1 103 Figure 4.17 Representative slices from the 13C and 15N-edited NOESY 105 Figure 4.18 Constant-time 13C-1H HSQC of 13C, 15N-labeled Ca2+-free DdCAD-1 in aromatic region 107 Figure 4.19 Combined 13C /13C chemical shift index plot of Ca2+-free DdCAD-1 110 Figure 4.20 Combined 13C /13C chemical shift index plot of Ca2+-bound DdCAD-1 111 Figure 4.21 A representative slice from 3D 15N-edited NOESY recorded on Ca2+-free DdCAD-1 113 Figure 4.22 Representative slices from 4D NOESY spectra recorded on Ca2+-free DdCAD-1 114 Figure 4.23 Sequential- and medium- range NOE pattern of Ca2+-free DdCAD-1 115 Figure 4.24 Sequential- and medium- range NOE pattern of Ca2+-bound DdCAD-1 116 Figure 4.25 Solution structures of 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H i - HNi H i - HNi H i - HNi+1 H i - HNi+1 H i - HNi+1 H i - HNi-1 H i - HNi-1 Hi-H i H i - H 1i H i - H 2i Hi-H i H i - H 1i H i - H 2i Hi-H i H i - HNi H i - HNi H i - HNi+1 H i - HNi+1 H i - HNi-1 H i - HNi-1 Hi-H i H i - H 1i H i - H 2i Hi-H i H i - H 1i H i - H 2i Hi-H i H 2i - HNi H 1i - HNi H 2i - HNi+1 H 1i - HNi+1 5018 5018 4765 4765 4765 4726 4726 5018 5018 5018 5018 5018 5018 5018 4517 4517 4260 4260 4279 4279 4517 4517 4517 4517 4517 4517 4517 2927 2927 2739 2739 Occurrence of H-H pairs within a certain distance