Crystal structure analysis of pils, a type lvb pilin from salmonella typhi

CRYSTAL STRUCTURE ANALYSIS OF PILS, A TYPE IVB PILIN FROM SALMONELLA TYPHI MANIKKOTH BALAKRISHNA ASHA NATIONAL UNIVERSITY OF SINGAPORE 2007 CRYSTAL STRUCTURE ANALYSIS OF PILS, A TYPE IVB PILIN FROM SALMONELLA TYPHI MANIKKOTH BALAKRISHNA ASHA (B.Sc., B.Ed., M.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS . This research work is by far one of the most significant scientific accomplishments in my life and it would have been impossible without the following people, who supported me and had belief in me. First and foremost, I want to express my wholehearted gratitude and deepest thanks to my mentor and research advisor Associate Professor K Swaminathan, for his invaluable support and guidance throughout my research work. He is not only a great scientist with deep vision but also, and most importantly, a kind and understanding person with a cheerful disposition. Especially, I would like to thank him for his patience during the writing of my thesis. I would also like to express my special and sincere thanks to Dr. Henry Mok Yu-Keung for initiating the project on the structure determination of PilS. I gratefully acknowledge the financial support rendered by the National University of Singapore in the form of Research Scholarship. I am also grateful to the academic and technical staffs at the Department of Biological Sciences who have helped me in one way or the other in my research work. I owe very special thanks to my colleagues Gayathri, Tien-Chye and especially Dileep and to all my friends at NUS. I want to thank them for all their help, support, interest and valuable hints. Also, I express my special word of thanks to Sivakumar (former graduate student of Dr. Swaminathan at IMCB) and Lissa for their help. I wish to express my sincere appreciation and thanks to Dr. Anand Saxena (Brookhaven National Laboratory, USA) for his great help in data collection. I convey my heartfelt thanks to Dr. Gerhard Gruber of School of Biological Sciences (SBS), Nanyang Technological University (NTU) for giving me an i opportunity to work in his lab as Research Associate even before the completion of my PhD. I also thank my friends at NTU. Above all I want to thank my family, which continuously supported me at all times. I thank my parents for teaching me the value of education at a young age and my uncle who instilled in me a desire for higher education. I wish to thank my parents for their love and support, especially at times when they looked after my son during my data collection trips. Also I am indebted to my brother Anil and sister Usha, and their families, whose ceaseless encouragement and unflinching support has helped me to shape my career and life. Words cannot express the love, encouragement and support I received from my husband Hari, without whose constant help and support, my Ph.D. research work would have remained a daydream and my dear sons, Bharat and Arjun whose smiles and love never let me forget what’s really important in life and buoyed me up. The loving family environment and support I enjoyed from all my family members was greatly instrumental in providing me the tranquility and enthusiasm to pursue my research with a piece of mind. ii PUBLICATION Parts of this thesis have already been or will be published in due course: Balakrishna, A. M., Tan, Y.Y., Mok, H,Y., Saxena, A.M. and Swaminathan, K. (2006). Crystallization and preliminary X-ray diffraction analysis of Salmonella typhi PilS. ACTA Cryst. F 62: 1024-1026. Crystal structure of Salmonella typhi PilS explains the structural basis of typhoid infection. Balakrishna, A. M., Mok, H,Y., Saxena, A.M. and Swaminathan, K. (in preparation). iii TABLE OF CONTENTS Page Acknowledgements i Publication iii Table of contents iv Summary viii List of abbreviations ix List of figures xii List of tables xiv CHAPTER MACROMOLECULAR X-RAY CRYSTALLOGRAPHY 1.1 CRYSTALLIZATION OF PROTEINS 1.2 BASIC CONCEPTS OF X-RAY CRYSTALLOGRAPHY 1.2.1 Crystal symmetry and unit-cell 1.2.2 Lattice and space group 1.3 X-RAY SOURCES AND DETECTORS 1.3.1 X-ray sources 1.3.2 X-ray detectors 1.4 DIFFRACTION OF X-RAYS BY A CRYSTAL 1.4.1 X-ray diffraction and Bragg’s law 1.4.2 The reciprocal lattice and Ewald sphere 1.5 10 DIFFRACTION DATA TO ELECTRON DENSITY 1.5.1 Structure Factor and electron density 11 iv 1.5.2 Fourier transform 13 1.5.3 Intensities and the phase problem 14 1.6 15 PROTEIN CRYSTAL STRUCTURE DETERMINATION 1.6.1 Direct method 15 1.6.2 Molecular replacement 15 1.6.3 Multiple isomorphous replacement 16 1.6.4 Multiple-wavelength anomalous dispersion 19 1.6.4.1 Anomalous scattering 19 1.6.4.2 Extracting phases from anomalous scattering data 21 1.7 22 TECHNIQUES FOR IMPROVEMENT OF ELECTRON DENSITY 1.7.1 Calculated structure factors 22 1.7.2 Solvent flattening 23 1.7.3 Molecular averaging 23 1.8 23 MAP FITTING AND REFINEMENT 1.8.1 Fitting of maps 23 1.8.2 Refinement of model coordinates 24 1.9 27 VALIDATION 1.9.1 The omit map 27 CHAPTER BIOLOGICAL BACKGROUND 2.1 BACTERIAL ADHESION 30 2.1.1 Fimbriae of Gram-negative bacteria 31 2.2 32 TYPE IV PILI 2.2.1 General secretion pathway of type IV pili 32 2.2.2 Type IV pilus functions 33 v 2.2.2.1 Surface motility 34 2.2.2.2 Microcolony and biofilm formation 35 2.2.2.3 Host-cell adhesion 36 2.2.2.4 Cell signaling 38 2.2.2.5 Apoptosis 38 2.2.2.6 DNA binding 39 2.2.3 Characteristics of type IV pili 39 2.2.4 The pilin structure 41 2.3 43 THE TYPE IVB PILI OF S. TYPHI 2.3.1 Role in pathogenesis 44 2.3.2 The receptor of S. typhi 46 2.3.3 Cystic fibrosis and typhoid fever 48 2.4 49 THE PURPOSE OF THIS STUDY CHAPTER MATERIALS AND METHODS 3.1 EXPRESSION AND PURIFICATION OF RECOMBINANT PILS 50 3.2 BIOPHYSICAL CHARACTERIZATION 52 3.2.1 Dynamic light scattering 52 3.2.2 Mass spectrometry 53 3.3 CRYSTALLIZATION 54 3.4 DATA COLLECTION AND PROCESSING 55 3.4.1 Molecular symmetry 56 3.5 56 MODEL BUILDING AND REFINEMENT 3.5.1 Selenium position determination 56 3.5.2 Structure refinement of ∆PilS 58 vi 3.6 ∆PILS-PEPTIDE COMPLEX AND REDUCED ∆PILS STRUCTURES 58 3.6.1 Crystallization 58 3.6.2 Data collection 59 3.6.3 Structure analysis and refinement 59 CHAPTER RESULTS AND DISCUSSION 4.1 THREE-DIMENSIONAL STRUCTURE OF TYPE IVB PILIN 61 4.1.1 Structure determination 61 4.1.2 Overall structure of ∆PilS 61 4.2 STRUCTURAL COMPARISON OF TYPE IVB PILINS 65 4.3 INSIGHTS INTO THE PEPTIDE BINDING POCKET 71 4.3.1 ∆PilS-CFTR peptide complex crystallization 72 4.3.2 The complex structure 73 4.3.3 The peptide binding surface of ∆PilS 75 4.4 79 REDUCED STRUCTURE 4.4.1 Structural overview 79 4.4.2 The role of disulfide bonds 81 4.5 DISCUSSION 86 4.6 FUTURE DIRECTIONS 88 4.7 CONCLUDING REMARKS 88 REFERENCES 90 vii SUMMARY This is a report on the structure determination of the PilS dimer by X-ray crystallography. The recombinant protein from Salmonella typhi was overexpressed, purified and crystallized. The crystals belong to space group P21212, with unit-cell parameters a = 77.88, b = 114.53 and c = 31.75 Å. The selenomethionine derivative of the PilS protein was overexpressed, purified and crystallized in the same space group. Data sets for the selenomethionine derivative crystal have been collected to 2.1 Å resolution using synchrotron radiation for multiwavelength anomalous dispersion (MAD) phasing. Understanding of the subunit structure and assembly architecture that produce the Salmonella typhi pili filaments is crucial for understanding pilus functions and for designing vaccines and therapeutics that are directed to blocking pilus activities. The target receptor for the S. typhi pilus is a stretch of 10 residues from the first extracellular domain of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (Tsui et al., 2003). The structure of the 26 N-terminal amino acid truncated Type IVb structural pilin monomer (∆PilS) from S. typhi was determined by NMR (Xu et al., 2004). In the present study, this ∆PilS protein has been crystallized by the sitting drop vapor diffusion method. The structure of this protein is determined by the multiwavelength anomalous dispersion (MAD) method. The complex-∆PilS crystal structure with the CFTR peptide has given us further insight into the potential residues that are essential for receptor binding and the implications of the disulfide bond in pilus assembly. viii is observed in the loop region (residues 82-86) which lies in between the α helix and β strand. (Fig. 4-9). The fact that the peptide is present at the dimer interface of the ∆PilS structure, and that the conformation of the Lysine residues (LysA75 and LysB120) from the main chain protein which interacts with the CFTR peptide is altered in the complex structure, shows that the interaction is specific. Study by Pier et al has shown that residue 113-117 (NKEER) of CFTR are very essential for the S. typhi pilus binding and in our complex structure we observe that E115 & E116 (Glu8 & Glu9 respectively) are involved in binding to PilS protein. Preliminary Isothermal titration calorimetry (ITC) experiments of the 10-mer CFTR peptide (108-117) with ∆PilS protein did show that there is some binding (results not shown). Even in the NMR binding studies of PilS, the authors have concluded that complete binding site could be comprised of charged residues from different subunits [Xu et al., 2004]. It may be possible that this is an intermediatory stage of binding. Further binding and mutational studies have to be carried out to confirm this. The D-region in both type IVa and IVb pilins is stabilized by a conserved disulfide bond, which links the D-region to the α-helix/β- sheet scaffold. From our structure we observe that Lys30 (which lies on α1) interacts with Glu162 of the neighboring subunit that lies next to Cys163, which forms a disulfide bond with Cys126. We propose that if the disulfide bond between the two conserved cysteine residues is affected, it will displace the two chains, and can no longer form conducive salt bridges with neighboring pilin molecules thereby disrupting the interaction between subunits and pilus formation. However, our reduced structure breaks the disulfide bond only partially. The S–S distance is about 2.5 Å in each monomer in the asymmetric unit. These distances are intermediate between the 2.03 Å that is expected for a fully oxidized disulfide and 87 3.7 Å that one might expect for a fully reduced disulfide. One explanation of the intermediate S–S distances observed in our structure is that they are the time and space-averaged results of a mixture of oxidized and reduced disulfides. 4.6 FUTURE DIRECTIONS The binding site of CFTR peptide to that of the ∆PilS need to be further confirmed by doing point mutations of Lys 75 and Lys 120, which are shown to interact with the predominantly negatively charged 10-mer peptide of the CFTR. We are constructing Lys75Ala and Lys120Ala mutants using the GeneTailor SiteDirected Mutagenesis System to confirm the receptor binding sites of the ∆PilS protein. Once we are able to successfully express these mutants, we plan to further binding studies using Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). The aim of reduction was to see if there is any structural change when the disulphide is broken, but we don’t see any such changes in our reduced structure. Therefore in order to clearly understand the implications of the disulfide bond in the ∆PilS structure a Cys→Ser double mutant of the protein has to be crystallized. Currently we are constructing the cysteine mutants also using the GeneTailor SiteDirected Mutagenesis System. If the mutants get crystallized, then the resulting structure can give a better understanding of the implications of the disulfide formation in the type IVb pilins, through better comparison of the reduced structure with the current oxidized ∆PilS structure. This will ultimately shed light on the structural role of the D region in type IV pilins. 4.7 CONCLUDING REMARKS 88 In this study, we have solved the crystal structure of ∆PilS pilin to look into the features in the receptor-binding site and have explored the role of disulfide in the pilus formation. 89 REFERENCES Aas, F.E., Lovold, C. and Koomey, M. (2002). 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Immun. 68, 3067-3073. 102 [...]... to a supersaturated state is indispensable for crystallization To achieve usable crystal growth, the supersaturation must be properly regulated Maintaining a high supersaturation would result in the formation of too many nuclei and therefore too many small crystals 2 1.2 BASIC CONCEPTS OF X-RAY CRYSTALLOGRAPHY 1.2.1 Crystal symmetry and unit-cell Crystals exhibit clear-cut faces and edges that are... its variations)] in the 7 crystal systems allow a total of 14 Bravais lattices in crystallography The combination of the lattice type of a crystal system and the applicable symmetry elements for that system (including the screw axis that degenerates from rotation and the glide plane that degenerates from reflection) will define the entire packing pattern of molecules, known as space group, for that system... collection and the wavelength, the unit-cell parameters can be determined As the reciprocal lattice bears a direct relationship with the crystal, rotation of the crystal will cause a similar rotation of the reciprocal lattice A geometrical description of diffraction that encompasses Bragg's law was originally proposed by Ewald The advantage of this description, the Ewald construction, is that it allows... of target material at the edge of the focal spot The heating of the anode caused by the electron beam at the focal spot limits the maximum power of the tube This limit is reduced in a rotating anode X-ray generator, where the anode is a rotating cylinder instead of a fixed piece of metal The rotating target can sustain 7-45 times more power loading than sealed tubes The second advantage of the rotating... structure of biological macromolecules The major rate determining step in protein crystallography is the crystallization process 1.1 CRYSTALLIZATION OF PROTEINS The process of crystallization of a macromolecule is very complex Growth of a protein crystal starts from a supersaturated solution of the macromolecule, and evolves towards a thermodynamically stable state in which the protein is partitioned... observer to calculate which Bragg peaks will be observable if the orientation of the crystal on the goniostat is known As an example, consider a two-dimensional reciprocal lattice Constructive interference occurs when a set of crystal lattice planes separated by a spacing of dhkl are inclined to an angle θhkl with respect to the incident beam A diffracted beam can be measured at an angle 2θhkl from the... 1.5 DIFFRACTION DATA TO ELECTRON DENSITY The outcome of X-ray data collection is a list of intensities of all observed diffraction maxima, hkl The observed diffraction pattern and the electron density distribution within a unit-cell (and hence the crystal) are the Fourier transformations of each other, which means that we can convert the crystallographic data into an arrangement of atoms within a unit-cell... shorter wavelength has lower absorption along its path and in the crystal Synchrotron radiation, in contrast to X-ray tube radiation, is highly polarized The polarization of 5 the X-ray beam from a synchrotron has an effect on the anomalous X-ray scattering of atoms which occurs when the X-ray wavelength approaches the absorption edge wavelength 1.3.2 X-ray detectors In an X-ray diffraction experiment... similarity of diffraction to ordinary reflection and deduced a simple equation treating diffraction as “reflection” from planes in the lattice In order to derive the equation, we consider an X-ray beam that is incident on a pair of parallel planes P1 and P2 with interplanar spacing d The parallel incident rays 1 and 2 make an angle θ with these planes Electrons located at O and C will be forced to vibrate... observation that in a diffraction experiment, the diffraction maximum of a set of planes with finer interplanar spacing is recorded farther from the direct beam position than that for a set of planes with greater interplanar spacing C Figure 1-3 Bragg’s law 8 By rearranging Bragg’s law, sin θ = nλ/2 (1/d), and thus sin θ is inversely proportional to d, the interplanar spacing in the crystal lattice . course: Balakrishna, A. M., Tan, Y.Y., Mok, H,Y., Saxena, A. M. and Swaminathan, K. (2006). Crystallization and preliminary X-ray diffraction analysis of Salmonella typhi PilS. ACTA Cryst. F 62:. CRYSTAL STRUCTURE ANALYSIS OF PILS, A TYPE IVB PILIN FROM SALMONELLA TYPHI MANIKKOTH BALAKRISHNA ASHA NATIONAL UNIVERSITY OF SINGAPORE. 1024-1026. Crystal structure of Salmonella typhi PilS explains the structural basis of typhoid infection. Balakrishna, A. M., Mok, H,Y., Saxena, A. M. and Swaminathan, K. (in preparation).