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NMR structural studies on the pilin monomer PilS from Salmonella typhi XU XINGFU A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 i ACKNOWLEDGEMENTS I would like to express my heartfelt appreciation and gratitude to my supervisor Dr Yu-Keung Mok, Henry for his patience, encouragement and guidance during the course of the project I would like to thank Dr Yang Daiwen for the technical support for my NMR experiments and for stimulating discussion on my project Special thanks go out to Mr Li Kai and Mr Zheng Yu for their useful scripts Many thanks to post-doctors and students from NMR structural biology lab and my friends in Department of Biological Sciences and other departments or institutes, who made me feel so much at home and made my stay in NUS a pleasant learning experience Finally, I wish to thank The National University of Singapore for granting me a Research Scholarship i Contents ACKNOWLEDGEMENTS i Contents ii List of figures .v List of tables .vii Abbreviations viii Summary x Introduction - 1.1 Introduction to biomolecular NMR - 1.2 Type IV pilins and their structures - 1.3 The aim of this study - 18 - Protein expression and purification - 19 2.1 Introduction - 19 2.2 Materials and methods .- 20 2.2.1 M9 medium .- 20 2.2.2 Preparation of competent E coli cells .- 20 2.2.3 Transformation of competent cells - 20 2.2.4 Expression system .- 21 2.2.5 Protein expression - 21 2.2.5.1 Determination of target protein solubility - 21 2.2.5.2 Protein expression - 22 2.2.6 Protein purification - 22 2.2.6.1 Pre-column treatment .- 22 2.2.6.2 Ni-NTA affinity chromatography - 23 2.2.6.3 Protein refolding - 23 2.2.6.4 Thrombin cleavage and gel filtration - 24 2.2.7 One dimensional 1H NMR experiment for the unlabelled sample - 24 2.2.8 Stable-isotopic labeling of PilS24 .- 25 2.2.8.1 15N uniformly labeled sample .- 25 2.2.8.2 15N-13C uniformly labeled sample and 10% 13C labeled sample - 25 2.2.9 Measurement of protein concentration in solution .- 25 2.3 Result - 26 2.3.1 PilS24 was expressed as inclusion bodies - 26 2.3.2 Protein purification - 27 2.3.2.1 Ni-NTA affinity chromatography - 27 2.3.2.2 Protein refolding and thrombin cleavage - 28 2.3.2.3 Gel filtration - 28 2.3.2.4 The successful refolding of PilS24 - 29 2.3.2.5 The final construct of protein sample .- 30 - ii Discussion - 30 2.4.1 Expression and purification system - 30 2.4.2 Protein refolding .- 32 2.4.3 Sample labeling - 33 2.4.4 Characterization of refolding by one dimensional detected proton NMR - 34 - 1H, 15N, 13C assignments and secondary structure characterization of PilS24 - 36 3.1 Introduction - 36 3.2 Materials and methods .- 38 3.2.1 NMR experiments .- 38 3.2.1.1 2D 1H-15N HSQC spectrum - 38 3.2.1.2 HNCACB and CBCA(CO)NH .- 38 3.2.1.3 HNCO and HN(CA)CO - 40 3.2.1.4 C(CO)NH and H(CO)NH .- 40 3.2.1.5 HCCH-TOCSY .- 41 3.2.1.6 1H-13C CT-HSQC and 1H-13C HSQC .- 42 3.2.1.7 HNHA experiment - 42 3.2.1.8 H/D exchange measurements - 43 3.2.1.9 15N edited NOESY .- 43 3.2.1.10 13C edited NOESY .- 44 3.2.2 Chemical shift assignment .- 44 3.2.2.1 Backbone sequential assignment - 44 3.2.2.2 Aliphatic side chain assignment and stereospecific assignment .- 45 3.2.3 Secondary structure characterizations .- 45 3.2.3.1 Chemical shift index prediction and 3JHNHα coupling constant - 45 3.2.3.2 Sequential NOE pattern and short, medium-range NOE analysis and hydrogen bond analysis - 46 3.3 Results and discussions .- 46 3.3.1 Backbone assignment and aliphatic side chain assignment .- 46 3.3.1.1 Backbone assignment .- 46 3.3.1.2 Aliphatic side chain assignment - 49 3.3.1.3 Stereo-specific assignment of methyl groups of leucine and valine .- 52 3.3.2 Secondary structure identification .- 54 3.3.2.1 Chemical shift index .- 54 3.3.2.2 NOE analysis - 54 3.3.2.3 Hydrogen bond analysis - 55 3.3.2.4 J coupling constant analysis - 59 3.4 Conclusion - 60 - The three dimensional solution structure of PilS24 - 62 4.1 Introduction - 62 4.2 Materials and methods .- 63 4.2.1 NOE assignment of 15N-edited NOESY and 13C-edited NOESY - 63 4.2.2 Structure calculations - 63 4.2.2.1 NOE restraints - 63 4.2.2.2 Dihedral angle restraints .- 64 - iii 4.2.2.3 Hydrogen bond restraints and disulphide bond restraint - 64 4.2.2.4 Structure calculation, energy minimization and statistics .- 64 4.3 Results - 65 4.3.1 Assignment of NOE - 65 4.3.2 Structural statistics - 66 4.4 Discussions - 67 4.4.1 Description of the structure of PilS24 .- 67 4.4.2 Full length structure of PilS .- 70 4.4.3 Helices .- 70 4.4.4 β–sheet .- 71 4.4.5 Unstructured regions and loops - 71 4.4.6 Hydrophobic Core .- 71 4.4.7 Proline conformation - 72 4.4.8 Possible residues for binding CFTR - 73 4.4.9 Comparison to other pilin structures - 76 - Conclusions and future work - 79 References - 81 - iv List of figures FIGURE 1.1 Sequence alignment of Type IV prepilins (Clustalw 1.82) FIGURE 1.2 Type IVa pilin structures 12 FIGURE 1.3 Three-layer representation of the modeled N gonorrhoeae pilus fiber 13 FIGURE 1.4 Structure of Type IVb pilin from V cholerae 15 FIGURE Side view of the structure-based TCP model 16 FIGURE 2.1 PilS was expressed as inclusion bodies 26 FIGURE 2.2 Ni-NTA affinity purification 27 FIGURE 2.3 Gel filtration column purification 28 FIGURE 2.4 Expression and purification of 15N, 13C labeled PilS24 29 FIGURE 2.5 1-D Proton NMR spectrum for unlabeled PilS24 30 FIGURE 3.1 CA, CB and CO connectivity for a stretch of residues from A58 47 to G65 FIGURE 3.2 The 1H-15N HSQC spectrum of 15N labeled PilS24 48 FIGURE 3.3 Aliphatic side chain assignments 49 FIGURE 3.4 Selected 1H(F3) and 1H(F1) planes at different 13C(F2) chemical 50 shifts of the HCCH-TOCSY spectrum illustrating connectivity FIGURE 3.5 Methyl regions from 1H-13C- CT-HSQC spectrum 51 FIGURE 3.6 13 52 C-13C scarlar coupling fine structure for every type of amino-acid methyl group FIGURE 3.7 H-13C methyl region of 1H-13C HSQC spectrum of 10% v 13 C 53 labeled PilS24 FIGURE 3.8 Combined 13Cα/13Cβ chemical shift index plot of PilS24 FIGURE 3.9 The 55 and 57 Hydrogen bond restraints from amide proton exchange 58 secondary structure analysis based on short- medium-NOEs and 3JHNHα coupling constant FIGURE 3.10 experiments FIGURE 3.11 Inter-strand NH-NH, NH-Hα NOEs and hydrogen bonds of 59 PilS24 FIGURE 3.12 Karplus curve and its relation to 3JHNHα 60 FIGURE 4.1 Stereo-view shows the superposition of the backbone atoms for 68 the 10 solution structures of PilS24 FIGURE 4.2 Ribbon diagram representation of PilS24 69 FIGURE 4.3 Hydrophobic core formed by helix and strands 3, 4, 72 FIGURE 4.4 A model of PilS pilus assembly 74 FIGURE 4.5 Molecular surface of PilS24 75 FIGURE 4.6 Comparison of type IV pilin structures from secondary structure 78 diagrams vi List of tables Table 4.1 Summary of the restraints used to calculate the structures 66 Table 4.2 Structural Statistics for the CYANA calculation and final ensemble 67 Table 4.3 Angles between helices 70 vii Abbreviations 1D One-dimensional 2D Two-dimensional 3D Three-dimensional a.a Amino acid COSY Correlated spectroscopy E coli Escherichia coli EDTA Ethylenediamine tetraacetic acid F1 The acquired frequency dimension in an NMR spectrum F2/F3 Indirectly detected frequency dimension in an NMR spectrum FID Free induction decay HSQC Heteronuclear single quantum correlation spectrum IPTG Isopropyl β-D-thiogalactoside NMR Nuclear magnetic resonance NOE Nuclear Overhauser effect NOESY Nuclear Overhauser enhanced spectroscopy OD Optical density ppm Parts per million rms Root mean square SDS Sodium dodecyl sulphate TOCSY Total correlation spectroscopy viii Tris 2-amino-2-(hydroxymethyl-1,3-propanediol Ala, A alanine Arg, R arginine Asn, N asparagine Asp, D aspartic acid Cys, C cysteine Glu, G glycine His, H histidine Ile, I isoleucine Leu, L leucine Lys, K lysine Met, M methionine Phe, F phenylalanine Pro, P proline Ser, S serine Thr, T threonine Trp, W tryptophan Tyr, Y tyrosine Val, V valine ix FIGURE 4.5 Molecular surface of PilS24 The possible residues for binding to CFTR peptide are shown (calculated and displayed by MOLMOL) - 75 - 4.4.9 Comparison to other pilin structures PilS has a very similar folding topology compared to that of TCP pilin Both of them have an extended N-terminal α–helix which is packed against anti-parallel β–strands to form the hydrophobic core of the protein The disulphide bonded D region in PilS24 is similar to TCP pilin by having a pair of α-helices located on top of two shorter anti-parallel β-strands The loop between N-terminal α-helix and the first β-strand is termed αβ loop in TCP pilin This αβ loop can also be found in truncated PilS However, there are still some distinct differences between these type IVb structures There are two extra strands at the edge of the β-sheet in PilS, while in TCP pilin the corresponding region is replaced with an extended loop of 18 residues The extra two strands are docked by the second helix and cause the sheet to twist in this edge The other difference lies on the length of disulphide bonded D-region The D-region of TCP pilin encompasses 65 residues and is the longest among Type IVb pilins PilS have the shortest D-regions among Type IVb pilins and consist of only 36 residues The extra length in the D-region of Tcp pilin is contributed by two extended loops between β3 and β4 (24 residues) and between β4 and α4 (12 residues) The corresponding regions in PilS between β5 and β6 and between β6 and α4 are only and residues in length respectively These two extended loops of Tcp pilin are highly exposed on the surface of the pilus and cover most of β-sheet at the opposite side of the N-terminal α-helix These loops also contain overlapping epitopes for protective TCP antibodies and encompass most of the functional domain residues - 76 - (Kirn, 2000) In contrast to Tcp pilin, these loops are relatively short in PilS and leaves most of the β-sheet opposite to the N-terminal helix exposed on the surface of the pilus A conserved architectural feature has been revealed by the Type IV pilin structures Comparison of the available Type IV pilin structures including MS 11 from N gonorrhoeae, PAK from P auroginosa, PilS from S typhi and TCP pilin revealed a conserved scaffold of a ladle shaped molecule that contain an extended N-terminal long α-helix wrapped by an anti-parallel β-sheet (Figure 4.6) The long α-helix is responsible for the polymerization of pilin molecules, while the anti-parallel β-sheet can be used to form the surface of cylindrical pilus The structural difference between Type IVb pilins and type IVa pilins lies on the αβ region and D region For the αβ region, Type IVb members contain a second helix which is perpendicular (or nearly perpendicular) to helix in orientation In contrast, the αβ region of type IVa member is composed of a minor 3-stranded sheet (PAK pilin) or a glycosylated loop (MS11) The deferences in D region of the two types of pilins are even more substantial In type IVb pilins, the segments between the two cysteines are longer in length and comprise two helices and two strands On the contrary, the disulfide bonded region of the type IVa pilins has the last strand of the major β-sheet linked to a peripheral C-terminal loop and delineates only a smaller segment The substantial difference between the two subgroups of pilin in the αβ and D-region could account for their differences in pilus assembly models of pili as well as the receptor binding specificity - 77 - FIGURE 4.6 Comparison of type IV pilin structures from secondary structure diagrams The region between the helix and major sheet is shown in yellow Disulfide bonded region is shown in green Disulfide bond is shown in violet A Tcp pilin from V cholerae (Type IVb) B PilS from S typhi (Type IVb) C MS11 from N gonorrhoeae (Type IVa) D PAK from P auroginosa (Type IVa) - 78 - Conclusions and future work Conclusions This study aimed to solve the type IVb pilin PilS structure from Salmonella typhi using NMR The solved structure will provide structural information for rational design of drugs or vaccines for the prevention and treatment of human typhoid fever The N-terminal truncated PilS was expressed in E coli and purified by Ni-affinity chromatography, refolded and further purified using gel filtration chromatography The same expression system and purification steps were employed to produce isotope labeled sample for the NMR studies Near complete 1H, 13 C and 15 N backbone and side chain chemical shift assignments were obtained using a suite of 3-D NMR experiments The chemical shift assignment provided important information for the later NMR structural studies of PilS The Secondary structure analysis based on Chemical Shift Index, short and medium NOE analysis, J coupling constant, and hydrogen bonds could define that PilS is composed of helices and strands The three dimensional structure of PilS calculated shows an α/β roll fold similar to the recently determined X-ray structure of pilin from Vibrio cholera toxin-coregulated pilus (TCP) The conserved and extended N-terminal helix of both proteins packed against an anti-parallel β-sheet to form the hydrophobic core of the protein Both proteins contain an N-terminal αβ-loop and a C-terminal - 79 - disulphide-bonded region (D-region) at opposite edges of the β-sheet There are, however, distinct differences between structures of the two proteins PilS has an extra-pair of β-strands in the αβ loop and its D-region is only half the length (36 residues) of that of TCP pilin (65 residues) Comparison of all type IV lipin structures suggested that a conserved scaffold exists among type IV pilins even though they have distinct amino acid sequences Future work The refinement of PilS structure can be done by completing the aromatic ring proton assignment More long range NOEs can be assigned once it is completed The structure of PilS and the tentative model of pilus provided the clues on possible charged residues that are involved in the binding CFTR peptide Site-directed mutants can be designed to check whether these residues are indeed involved in pilus function - 80 - References Bieber, D et al (1998) Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli Science 280: 2114-2117 Bloch, F (1946) Phys Rev 70:460 Bradley, D.E (1980) A function of Pseudomonas aeruginosa PAO polar pili: twitching motility Can J Microbiol 26: 146–154 Campbell, A.P et al (1997) Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study Biochemistry 36: 12791-12801 Case, D.A et al (2002) Amber 7, University of California, San Francisco Cavanagh et al (1996) Protein NMR Spectroscopy, Academic Press, San Diego, USA, Chiang, S.L., Taylor, R.K., Koomey, M., and Mekalanos, J.J (1995) Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance Mol Microbiol 17: 1133–1142 Clore, G.M., Gronenborn, A.M (2000) Protein NMR spectroscopy in structural genomics Nat Struct Biol Suppl: 982-985 Collinson, S K., Emody, L., Muller, K.-H., Trust, T J., and Kay, W W (1991) Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis J Bacteriol 173:4773–4781 - 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89 - [...]... operon was cloned and identified as a type IVb operon (Zhang, 1997) Among the 11 genes of this operon, PilS encodes the prepilin structural protein Further studies suggest that a PilS mutant of S typhi entered human intestinal INT407 cells in culture to levels only 525% of those of the wild-type strain The inhibition experiments also indicate that the entry of S typhi into epithelial cells is strongly... the sequential connectivity by linking spin systems together The second step is to assign NOE peaks to proton pairs based on the previous chemical shift assignment NOE assignment can then be converted to distance constraints between protons The last step is structural calculation by computational techniques (distance geometry and simulated annealing) using the obtained constraints Two dimensional NMR. .. in the proposed fiber model is comprised of residues 123 to 143 and 152 to 158, as well as the disaccharide at Ser63, from each monomer The central layer is a continuous 25-stranded β-sheet, made up of the four strands from the antiparallel β-sheet as well as the sugar loop from each of the five pilin monomers present in each turn The innermost layer is a parallel coiled-coil made up of the highly conserved... located in the cytoplasm or associated with the cytoplasmic face of the inner membrane that may energize secretion by ATP hydrolysis and (iv) an -8- Figure 1.1 Sequence alignment of Type IV prepilins (Clustalw 1.82) (Thompson, 1994) R64: Prepilin from Plasmid R64 Type IVb pilus PilS: Prepilin from Salmonella typhi Type IVb pilus GC: Prepilin from Nesseria gonorrhoeae MS11 Type IVa pilus PAK: Prepilin from. .. epithelial cell Soluble prePilS protein inhibits the entry of S typhi into intestinal epithelial cell and this inhibition effect can be neutralized by peptide corresponding to the first extracellular domain of CFTR but not a scrambled one (Zhang, 2000) 1.3 The aim of this study To elucidate the molecular fold and architecture of the virulence factor PilS from Salmonella typhi will provide a foundation... foundation for the understanding of host-pathogen interaction of human typhoid fever The detailed structural information of this virulence factor can also aid the rational design of bacterial vaccines and therapeutic agents capable of inhibiting pilus adhesion on host cells - 18 - 2 Protein expression and purification 2.1 Introduction From the earliest NMR exploration of proteins it is realized that resonance... (B) The outermost hypervariable region consists of a.a 123–143 and a.a 152–158 as well as the disaccharide at Ser63 (C) End -on view of the central β-sheet layer (D) Side view of the central layer, showing the continuous β-strand hydrogen-bonding interactions around the fiber (E) The end-view of the central coiled-coil layer shows that the center of the fiber is quite closely packed (F) Side view of the. .. are responsible for recognizing and binding to specific receptor on host cells The binding event may activate signal transduction cascade in the host cell that has diverse consequences including activation of innate host defenses or the subversion of cellular processes facilitating bacterial colonization and invasion In addition to the binding event, many others events also activate the expression of... residues at the C-terminal region The C-terminal amino acid (glycine) of signal peptides and the 5th amino acid of mature pilin are conserved among Type IV prepilins Type IV pili are usually divided into two groups Group IVa consists of pili from P aeruginosa, N gonorrhoeae, M.bovis, and so on They are closely related in amino acid sequence and produced from prepilin molecules through the cleavage... protons that a protein contains will make the 1D proton spectrum extremely overlapped and largely interpretable The application of 2D spectra makes it possible to study small proteins by NMR In two-dimensional NMR spectroscopy the second dimension is another time domain which can be established by introducing another pulse and systemically increasing the time between the two pulses The strategy for protein ... (Thompson, 1994) R64: Prepilin from Plasmid R64 Type IVb pilus PilS: Prepilin from Salmonella typhi Type IVb pilus GC: Prepilin from Nesseria gonorrhoeae MS11 Type IVa pilus PAK: Prepilin from. .. Comparison of the pilin structures of these two subclasses (Type IVa and IVb) suggested that Type IV pilins share a conserved architectural scaffold - 16 - PilS from Salmonella typhi Salmonella. .. Pil operon was cloned and identified as a type IVb operon (Zhang, 1997) Among the 11 genes of this operon, PilS encodes the prepilin structural protein Further studies suggest that a PilS mutant