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• W2021862499 abstract "Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal α-helix. Engineering of a truncated pilin fromPseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an α-helix at the N terminus that lies across a 4-stranded antiparallel β-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction. Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal α-helix. Engineering of a truncated pilin fromPseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an α-helix at the N terminus that lies across a 4-stranded antiparallel β-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction. Pseudomonas aeruginosa strain K double-quantum-filtered correlated spectroscopy heteronuclear single quantum coherence Luria broth Neisseria gonorrhoeae strain MS-11 nuclear Overhauser enhancement spectroscopy P. aeruginosa strain O phosphate-buffered saline root mean square total correlation spectroscopy 2-acetamido-2-deoxy-β-d- galactopyranosyl-β-d-galactopyranoside gangliotetraosyl ceramide gangliotriaosyl ceramide Pseudomonas aeruginosa is a common, rod-shaped, Gram-negative bacterium that is an opportunistic pathogen and frequently causes life-threatening infections in burn, cancer, cystic fibrosis, immuno-compromised, and intensive care patients (1Bodey G.P. Bolivar R. Fainstein V. Jadeja L. Rev. Infect. Dis. 1983; 5: 279-313Crossref PubMed Scopus (733) Google Scholar, 2Pier G.B. J. Infect. Dis. 1985; 151: 575-580Crossref PubMed Scopus (153) Google Scholar, 3Irvin R.T. Campa M. Bendinelli M. Friedman H. Pseudomonas aeruginosa as an Opportunistic Pathogen.in: Plenum Publishing Corp., New York1993: 19-42Crossref Google Scholar). The initial stage of Pseudomonas infection is the adherence of the pathogen to the mucosal cells of a susceptible host, which is mediated by a type IV pilus (2Pier G.B. J. Infect. Dis. 1985; 151: 575-580Crossref PubMed Scopus (153) Google Scholar, 3Irvin R.T. Campa M. Bendinelli M. Friedman H. Pseudomonas aeruginosa as an Opportunistic Pathogen.in: Plenum Publishing Corp., New York1993: 19-42Crossref Google Scholar, 4Paranchych W. Sastry P.A. Volpel K. Loh B.A. Speert D.P. Clin. Invest. Med. 1986; 9: 113-118PubMed Google Scholar, 5Lee K.K. Doig P. Irvin R.T. Paranchych W. Hodges R.S. Mol. Microbiol. 1989; 3: 1493-1499Crossref PubMed Scopus (48) Google Scholar). These type IV pili are produced by a variety of bacterial pathogens, including Pseudomonas,Neisseria, Moraxella, Dichelobacter, and Vibrio. While type IV pili are critical virulence factors, they also play a central role in twitching motility (6Bradley D.E. Can. J. Microbiol. 1980; 26: 146-154Crossref PubMed Scopus (242) Google Scholar), DNA transformation, and bacteriophage absorption (7Roncero C. Darzins A. Casadaban M.J. J. Bacteriol. 1990; 172: 1899-1904Crossref PubMed Google Scholar). Type IV pili are long fibers that extend from the bacterial surface and are composed of a single structural protein, pilin. These pili are ∼1,000–4,000 nm long, 5.2 nm in outer diameter (4Paranchych W. Sastry P.A. Volpel K. Loh B.A. Speert D.P. Clin. Invest. Med. 1986; 9: 113-118PubMed Google Scholar, 8Folkhard W.F. Marvin D.A. Watts T.H. Paranchych W. J. Mol. Biol. 1981; 149: 79-93Crossref PubMed Scopus (76) Google Scholar) and can be lengthened or retracted by assembly or disassembly of pilin subunits at the base of the pilus. The retraction of the pilus powers twitching motility and gliding motility (9Merz A.J. So M. Sheetz M.P. Nature. 2000; 407: 98-102Crossref PubMed Scopus (614) Google Scholar). Pilin is encoded by the pilA gene of the piloperon (10Mattick J.S. Whitchurch C.B. Alm R.A. Gene ( Amst .). 1996; 179: 147-155Crossref PubMed Scopus (114) Google Scholar), and is initially synthesized as a precursor, pre-pilin, which is cleaved, N-methylated, and assembled into a pilus. Each pilin protein contains a functional receptor-binding site; however, binding sites are only displayed at the tip of the pilus (11Lee K.K. Sheth H.B. Wong W.Y. Sherburne R. Paranchych W. Hodges R.S. Lingwood C.A. Krivan H. Irvin R.T. Mol. Microbiol. 1994; 11: 705-713Crossref PubMed Scopus (137) Google Scholar). This region is proposed to be the point of first contact between bacterial and host cells; consequently, pilus-mediated binding is considered a tip-associated event (11Lee K.K. Sheth H.B. Wong W.Y. Sherburne R. Paranchych W. Hodges R.S. Lingwood C.A. Krivan H. Irvin R.T. Mol. Microbiol. 1994; 11: 705-713Crossref PubMed Scopus (137) Google Scholar). Up to five pilin monomers are exposed at the tip of the pilus, resulting in multivalent receptor binding as is common for lectin-carbohydrate interactions (12Rini J.M. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 551-577Crossref PubMed Scopus (425) Google Scholar). The multivalency of the pilus and its variability in length have confounded the determination of accurate affinity constants for the pilus-cell surface interaction and prevented a comparison of the binding affinities of pili and synthetic receptor-binding domains. The C-terminal receptor-binding domain of pilin has been studied in detail for many different strains of P. aeruginosa. Extensive structural analysis of free peptides that form the C-terminal-binding domain has shown the presence of a type I β-turn followed by a type II β-turn (13Campbell A.P. McInnes C. Hodges R.S. Sykes B.D. Biochemistry. 1995; 34: 16255-16268Crossref PubMed Scopus (47) Google Scholar, 14Campbell A.P. Sheth H.B. Hodges R.S. Sykes B.D. Int. J. Pept. Protein Res. 1996; 48: 539-552Crossref PubMed Scopus (13) Google Scholar). Interestingly, theNeisseria gonorrhoeae strain MS-11 pilin contains two type I β-turns rather than a type I turn followed by a type II turn that is seen in PAK1 and PAO (13Campbell A.P. McInnes C. Hodges R.S. Sykes B.D. Biochemistry. 1995; 34: 16255-16268Crossref PubMed Scopus (47) Google Scholar, 15Parge H.E. Forest K.T. Hickey M.J. Christensen D.A. Getzoff E.D. Tainer J.A. Nature. 1995; 378: 32-38Crossref PubMed Scopus (398) Google Scholar, 16Hazes B. Sastry P.A. Hayakawa K. Read R.J. Irvin R.T. J. Mol. Biol. 2000; 299: 1005-1017Crossref PubMed Scopus (117) Google Scholar). The major host cell-surface receptors for the P. aeruginosapilin C-terminal receptor-binding domains are the common cell surface glycosphingolipids asialo-GM1 and asialo-GM2 (5Lee K.K. Doig P. Irvin R.T. Paranchych W. Hodges R.S. Mol. Microbiol. 1989; 3: 1493-1499Crossref PubMed Scopus (48) Google Scholar, 17Ramphal R. Sadoff J.C. Pyle M. Silipigni J.D. Infect. Immun. 1984; 44: 38-40Crossref PubMed Google Scholar, 18Ramphal R. Carnoy C. Fievre S. Michalski J.C. Houdret N. Lamblin G. Strecker G. Roussel P. Infect. Immun. 1991; 59: 700-704Crossref PubMed Google Scholar, 19Sheth H.B. Lee K.K. Paranchych W. Hodges R.S. Irvin R.T. Mol. Microbiol. 1994; 11: 715-723Crossref PubMed Scopus (113) Google Scholar, 20Yu L. Lee K.K. Hodges R.S. Paranchych W. Irvin R.T. Infect. Immun. 1994; 62: 5213-5219Crossref PubMed Google Scholar) which are up-regulated in susceptible patients. The minimal portion of the cell surface receptors asialo-GM1 and asialo-GM2recognized by the receptor-binding domain consists of the disaccharide βGalNAc(1–4)βGal (18Ramphal R. Carnoy C. Fievre S. Michalski J.C. Houdret N. Lamblin G. Strecker G. Roussel P. Infect. Immun. 1991; 59: 700-704Crossref PubMed Google Scholar, 19Sheth H.B. Lee K.K. Paranchych W. Hodges R.S. Irvin R.T. Mol. Microbiol. 1994; 11: 715-723Crossref PubMed Scopus (113) Google Scholar, 20Yu L. Lee K.K. Hodges R.S. Paranchych W. Irvin R.T. Infect. Immun. 1994; 62: 5213-5219Crossref PubMed Google Scholar). P. aeruginosa has both high innate resistance and a high frequency of acquired anti-microbial resistance (21Alonso A. Companario E. Martinez J.L. Microbiology. 1999; 145: 2857-2862Crossref PubMed Scopus (96) Google Scholar). Treatment ofP. aeruginosa infections is frequently problematic and associated with high morbidity and mortality rates in susceptible patient groups. Thus there is a significant interest in developing a vaccine against Pseudomonas. Antibodies have been raised against several proteins expressed by the bacteria including elastase, exotoxin A, and lipoprotein I (22Cryz S.J. Fürer E. Germanier R. Infect. Immun. 1983; 39: 1072-1079Crossref PubMed Google Scholar, 23Lydick E. McClean A.A. Woodhour A.F. Callahan L.T. J. Infect. Dis. 1985; 151: 375Crossref PubMed Scopus (6) Google Scholar, 24Finke M. Muth G. Reichhelm T. Thoma M. Duchene M. Hungerer K.D. Domdey H. von Specht B.U. Infect. Immun. 1991; 59: 1251-1254Crossref PubMed Google Scholar) as well as against various polysaccharides (25Pier G.B. Thomas D.M. J. Infect. Dis. 1983; 148: 206-213Crossref PubMed Scopus (19) Google Scholar). The C-terminal receptor-binding domain of pilin has been a natural target for vaccine development due to its early role in the attachment and infection process. The feasibility of using type IV pilus vaccines has been effectively demonstrated in both sheep and cattle where protection against Dichelobacter nodosus andMoraxella bovis, respectively, has been observed with pili-based vaccines (26Pugh G.W. Hughes D.E. Booth G.D. Am. J. Vet. Res. 1977; 38: 1519-1522PubMed Google Scholar, 27Farinha M.A. Conway B.D. Glasier L.M.G. Ellert N.W. Irvin R.T. Sherburne R. Paranchych W. Infect. Immun. 1994; 62: 4118-4123Crossref PubMed Google Scholar). A free peptide of this domain from PAK has been successfully used to produce monoclonal and polyclonal antibodies that confer protection in an animal infection model (28Sheth H.B. Glasier L.M. Ellert N.W. Cachia P. Kohn W. Lee K.K. Paranchych W. Hodges R.S. Irvin R.T. Biomedical Peptides Proteins Nucleic Acids. 1995; 1: 141-148PubMed Google Scholar). Synthetic peptide analogues based on this domain have been used to produce antibodies that show cross-reactivity between differentP. aeruginosa strains (29Cachia P.J. Glasier L.M. Hodgins R.R. Wong W.Y. Irvin R.T. Hodges R.S. J. Peptide Res. 1998; 52: 289-299Crossref PubMed Scopus (27) Google Scholar) and have been successfully used to generate vaccines. Herein, we describe the structure of a truncated, monomeric type IV pilin from P. aeruginosa strain K122-4 using NMR spectroscopy. The first 28 residues were truncated to prevent oligomerization. We demonstrate that this truncated protein retains the biological characteristics of the intact pilin monomer. This monomeric pilin is able to compete for receptor binding sites with a heterologous strain of Pseudomonas resulting in a significant decrease in mortality in an animal infection model. It follows, therefore, that the pilin monomer also retains the biological characteristics of the pilus fiber except for the oligomerization properties of the N-terminal 28 residues. We have developed a model for the formation of the pilus fiber based on electrostatic interactions between the globular portion of the pilin protein and previous x-ray diffraction data on pilus fibers (8Folkhard W.F. Marvin D.A. Watts T.H. Paranchych W. J. Mol. Biol. 1981; 149: 79-93Crossref PubMed Scopus (76) Google Scholar). Currently, this model is the best fit to the x-ray fiber diffraction data. The results presented here contribute significantly to our understanding of the structure and function of type IV pili and will aid in the development of novel therapeutic strategies for managing and preventing Pseudomonas infections. A DNA sequence encoding P. aeruginosa strain K122-4 pilin(29–150) was polymerase chain reaction-amplified from the full-length K122-4 pilin cDNA and cloned into the pRLD expression vector such that it had an in-frame OmpA leader sequence fused to the truncated pilin (30Tripet B., Yu, L. Bautista D.L. Wong W.Y. Irvin R.T. Hodges R.S. Protein Eng. 1996; 9: 1029-1042Crossref PubMed Scopus (81) Google Scholar) using standard techniques. Unlabeled and 15N labeled K122-4 pilin(29–150) was prepared from Escherichia coli DH5α cells, transformed with the pRLD plasmid carrying the K122-4 pilin(29–150) gene, grown in LB or minimal media containing 15NH4Cl. K122-4 pilin(29–150) was extracted from the periplasm by osmotic shock and purified by cation exchange with a CM-cellulose column (utilizing a linear gradient of 0–0.8 m NaCl) in 10 mm sodium acetate, pH 4.5. K122-4 pilin(29–150) was subsequently desalted on a Sephadex G75 column and lyophilized. The protein was deemed >95% pure by reverse phase high performance liquid chromatography analysis. The molecular weight of the sample was confirmed by electrospray mass spectroscopy using a Fisons VG Quattro mass spectrometer. The identity of the purified product was confirmed by N-terminal amino acid sequencing and immunoblotting using rabbit polyclonal anti-K122-4 pilus antisera. NMR analysis was performed on ∼0.5 mm K122-4 pilin(29–150) dissolved in either 90% H2O, 10% D2O or 99% D2O containing 20 mm deuterated sodium acetate, 1 mm NaN3, and 1 mm2,2-dimethyl-2-silapentane-5-sulfonic acid, pH 5.0. Sedimentation equilibrium analysis of K122-4 pilin(29–150) was performed with a Beckman XL-I analytical centrifuge with an AN50TI rotor at 20 °C. Data were collected using interference optics. Three protein concentrations were used: 0.75 mg ml−1, 2.03 mg ml−1 and 3.45 mg ml−1, each in 20 mm sodium phosphate, pH 7.2, 100 mm sodium chloride. The molecular mass of the truncated pilin was calculated from the protein concentration gradient at sedimentation equilibrium using a partial specific volume of 0.7255 ml g−1 as determined from the amino acid composition. Sedimentation equilibrium data was evaluated using a least-squares curve-fitting algorithm contained in the NonLin analysis program (31Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). NMR experiments were performed on Varian Unity 600 and INOVA 800 MHz spectrometers at 30 °C. Spectra were processed with NMRPipe (32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11533) Google Scholar) and analyzed using NMRView (33Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2677) Google Scholar).1H and 15N chemical shift assignments and may be found with BMRB accession number 4918 (34Keizer D.W. Kalisiak M. Crump M.P. Suh J.Y. Irvin R. Sykes B.D. J. Biomol. NMR. 2001; 19: 385-386Crossref PubMed Scopus (4) Google Scholar). An ensemble of 25 K122-4 pilin(29–150) structures was generated from 1032 distance restraints, 30 hydrogen-bond and 181 dihedral angle restraints (PDB code 1HPW) by using the dynamic simulated annealing protocols of Nilges et al. (35Nilges M. O'Donoghu S.I. Prog. Nuclear Mag. Res. Spectrosc. 1998; 32: 107-139Abstract Full Text PDF Scopus (224) Google Scholar) in the program X-PLOR version 3.8 (36Brünger A.T. X-PLOR Version 3.1 : A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1993Google Scholar). Interproton distance restraints were derived from a three-dimensional 15N-NOESY HSQC spectrum in H2O and a two-dimensional homonuclear NOESY spectra in D2O both with a τmix of 60 ms. NOEs were classified as strong, medium, or weak depending on their intensity. A list of NOE restraints used in structure calculations has been submitted to the PDB (code 1HPW). H-bonds were determined by observing a two-dimensional TOCSY spectrum collected 6 days after dissolving the protein sample in D2O buffer. 30 spin systems originating from backbone amide protons were observed and assigned as H-bonds after initial examination of ensembles of structures generated without incorporation of hydrogen bonds. φ Dihedral restraints were based on3 JHNHα coupling constants measured in a high resolution HNHA spectrum (37Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1050) Google Scholar). ψ angles were determined by analysis ofdNα/dαNratios but only incorporated into the regions of well defined secondary structure (38Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). Stereospecific assignments and χ1restraints were obtained from the analysis of the3 Jαβ coupling constants in DQF-COSY spectrum and the relative intensities of the NOEs from the NH and the Cα to Cβ protons in a 50-ms two-dimensional NOESY spectrum collected in D2O. All structure calculations included the disulfide bonds, Cys31-Cys67 and Cys103-Cys116 restrained to a distance of 2.02 ± 0.1 Å. No distance violations greater than 0.2 Å nor dihedral violations greater than 2° were found. All nonglycine residues in disallowed (φ, ψ) regions are located in the disordered termini of K122-4 pilin(29–150) (TableI).Table IExperimental restraints and structural statisticsNumber of Experimental restraintsDistance restraints from NOEs1032Intra (i =j)335Sequential (‖i −j‖ = 1)357Short (2 ≤ ‖i −j‖ ≤ 5)120Long (‖i −j‖ ≥ 6)220Hydrogen bond restraints30Dihedral angle restraints181Phi68Psi86Chi-127Total experimental restraints1213R.m.s. deviations from experimental dataNOEs0.0166 ± 0.002 ÅDihedrals0.39 ± 0.11 degreeR.m.s. deviations from ideal stereochemistryBonds0.00269 ± 0.00012 ÅAngles0.518 ± 0.011 degreeRamachandran analysisResidues in favored regions66%Residues in additional allowed regions28%Residues in generously allowed regions5%Residues in disallowed regions1% Open table in a new tab Pili from PAK were purified and biotinylated as described previously (20Yu L. Lee K.K. Hodges R.S. Paranchych W. Irvin R.T. Infect. Immun. 1994; 62: 5213-5219Crossref PubMed Google Scholar, 39Paranchych W. Sastry P.A. Frost L.S. Carpenter M. Armstrong G.D. Watts T.H. Can. J. Microbiol. 1979; 25: 1175-1181Crossref PubMed Scopus (37) Google Scholar). A polystyrene microtiter plate was coated with 50 μl of 40 μg ml−1asialo-GM1 in methanol. The solvent was evaporated at room temperature. Nonspecific binding sites were blocked by the addition of 200 μl per well of 5% (w/v) bovine serum albumin, in PBS buffer (150 mm NaCl, 10 mm sodium phosphate, pH 7.2). The plate was incubated at 37 °C for 1.5 h and the wells were then washed 3 times with 250 μl of 0.05% (w/v) bovine serum albumin in PBS buffer. 50-μl aliquots of biotinylated PAK pili (0.88 μg ml−1 in PBS buffer) containing various concentrations of the K122-4 pilin(29–150) were added to each well. The plate was incubated 2 h at 37 °C, washed (5 times with 250 μl of PBS buffer), followed by the addition of 50 μl/well of streptavidin-alkaline phosphatase conjugate at a 1:3000 dilution in PBS. The plate was then incubated for 1 h at room temperature, washed 5 times with 250 μl of PBS buffer, followed by the addition of 80 μl/well of the substrate solution (1 mg ml−1 p-nitrophenyl phosphate in 10% (v/v) diethanolamine, pH 9.8). Following a 10-min incubation at room temperature, microtiter plates were read at 405 nm. This study was performed in accordance with the Canadian Animal Care Guidelines and with the ethical approval of the University of Alberta Health Science Animal Welfare Committee. The A.BY/SnJ mice used in the study are a strain developed by Jackson Laboratory (Bar Harbor, ME) that are highly susceptible toPseudomonas infection (40Pennington J.E. Williams R.M. J. Infect. Dis. 1979; 139: 396-400Crossref PubMed Scopus (13) Google Scholar), with the LD50 for PAK being ∼3 × 105 colony forming units per mouse when challenged intraperitoneally (27Farinha M.A. Conway B.D. Glasier L.M.G. Ellert N.W. Irvin R.T. Sherburne R. Paranchych W. Infect. Immun. 1994; 62: 4118-4123Crossref PubMed Google Scholar). A.BY/SnJ mice were obtained from a breeding colony maintained behind barrier isolation. Mice were transferred from the breeding colony at 3 weeks of age and maintained in filtertop cages with a diet consisting of Purina PMI Certified Rodent Diet 5002 until they were ∼10 weeks of age and had a weight of 18–20 g. A double-blind study was then established where groups of 10 mice were administered, intraperitoneally, 100 μl of PBS, pH 7.2, containing either bovine serum albumin (400 μg) or K122-4 pilin(29–150) (100, 200, or 400 μg). Fifteen minutes later, mice were challenged with ∼5 times the LD50 of PAK in 100 μl of LB administered intraperitoneally as previously described (27Farinha M.A. Conway B.D. Glasier L.M.G. Ellert N.W. Irvin R.T. Sherburne R. Paranchych W. Infect. Immun. 1994; 62: 4118-4123Crossref PubMed Google Scholar, 28Sheth H.B. Glasier L.M. Ellert N.W. Cachia P. Kohn W. Lee K.K. Paranchych W. Hodges R.S. Irvin R.T. Biomedical Peptides Proteins Nucleic Acids. 1995; 1: 141-148PubMed Google Scholar). Mice were monitored hourly from 16 to 48 h post-challenge and euthanized when they displayed ruffled fur, evidence of dehydration, and had become non-responsive to stimuli. K122-4 pilin has significant homology to the pilin sequences from other bacterial species (Fig.1). The first 22 residues of pilin are highly conserved. These residues are highly apolar and extend from the rest of the protein. Consequently, they form an oligomerization domain in pilin. Pilin from P. aeruginosa strain K122-4 was engineered to exclude this oligomerization domain. The first 28 residues of the K122-4 pilin protein were therefore truncated to produce a protein that will be referred to herein as K122-4 pilin(29–150). The K122-4 pilin(29–150) was engineered to be exported to the periplasm by means of an OmpA leader sequence (30Tripet B., Yu, L. Bautista D.L. Wong W.Y. Irvin R.T. Hodges R.S. Protein Eng. 1996; 9: 1029-1042Crossref PubMed Scopus (81) Google Scholar). This protein was subsequently processed such that a soluble monomeric K122-4 pilin(29–150) with an additional 7 residues (Ala-Leu-Glu-Gly-Thr-Glu-Phe numbered 22–28 in this article), were fused to the N terminus of the K122-4 pilin(29–150) native sequence. The purified protein was subsequently analyzed by mass spectroscopy, analytical ultracentrifugation, and NMR. The mass of the purified protein was observed to be 13,107 Da by electrospray mass spectroscopy. Sedimentation equilibrium studies indicated a single homogeneous species with a molecular mass of 13,077 Da. Both of these values were in good agreement with the calculated molecular mass of the monomer (13,105 Da). A rotational correlation time of 7.4 ns was determined by NMR spectroscopic methods, which corresponds to a species of ∼14 kDa at 30 °C (41Suh J.Y. Spyracopoulos L. Keizer D.W. Irvin R.T. Sykes B.D. Biochemistry. 2001; 40: 3985-3995Crossref PubMed Scopus (18) Google Scholar). Taken together, these data indicate that K122-4 pilin(29–150) is monomeric up to the 0.5 mm concentrations used for NMR. To determine if the truncated form of the pilin protein from K122-4 was correctly folded and functional, two separate experiments were performed: binding of the truncated pilin to asialo-GM1, which requires a functional receptor-binding domain (the C-terminal loop), and protection of mice from P. aeruginosa infection by injection with the truncated K122-4 pilin(29–150). To determine if K122-4 pilin(29–150) retains receptor-binding function, a competitive inhibition assay was performed. The assay involved competitive binding between K122-4 pilin(29–150) and PAK pili (composed of the full-length PAK pilin protein assembled into pili) with the pili receptor, the naturally occurring membrane glycosphingolipid, asialo-GM1. PAK pili were chosen for the assay as they bind to the same receptors as K122-4 pili, are the best characterized and most readily purified pilus type, and are the most extensively studied pili from P. aeruginosa. The truncated K122-4 pilin(29–150)competitively inhibits PAK pili binding to immobilized asialo-GM1 in a dose-dependent manner (Fig.2 A). This suggests that the K122-4 pilin(29–150) monomer retains receptor-binding capability, and that the receptor-binding domain is intact in the truncated protein. To determine if K122-4 pilin(29–150) could protect mice against Pseudomonas infection, a double-blind study using A.BY/SnJ mice was carried out. Intraperitoneal administration of purified K122-4 pilin(29–150) in mice was found to delay and decrease mortality by infection with PAK (Fig. 2 B). The protection afforded by K122-4 pilin(29–150) appears to be dose-dependent, with both the 200 and 400 μg of dose of K122-4 pilin(29–150) conferring substantial protection against infection for over 35 h. The structure of K122-4 pilin(29–150) was determined using NMR spectroscopy (Fig.3). The C-terminal portion of the protein folds into a four-stranded antiparallel β-sheet structure composed of residues 78–87, 91–100, 110–119, and 126–133. This β-sheet structure folds around an α-helix that is comprised of residues near the N terminus (31–54). The α-helix lies at approximately a 45° angle from the axis of the β-sheet (Fig. 3 B). Most of the hydrophobic residues point into the center of the molecule anchoring the helix across the β-sheet. The outer surface of the molecule is composed primarily of polar residues. The hydrophobic interface between the helix and the four β-strands, as well as the loops between them, is comprised of residues Leu33, Leu39, Leu43, Val47, Ile50, and Phe51 of the α-helix with residues Val81, Ala87, Ile95, Ala97, Leu113, Leu115, Leu117, Trp127, and Leu138. These residues, for the most part, appear to be conserved (Fig. 1). The secondary structural elements constitute a well defined bundle with r.m.s. distributions about the mean coordinate positions of 0.71 ± 0.18 Å for backbone atoms, and 1.08 ± 0.17 Å for all heavy atoms. The loop structures (residues 28–30, 55–77, 88–90, 101–109, 120–125, and 134–150) connecting the α-helix and β-strands are not as well defined. Except for residues 76 and 77, these residues tend to display random coil amide 1HN and 15N chemical shifts and high r.m.s. deviations within the ensemble of calculated structures. The first 7 residues of K122-4 pilin(29–150), that precede the actual protein sequence starting at residue 29, are completely disordered and thus will not be discussed further. The C-terminal receptor-binding domain of K122-4 pilin is composed of residues Ala128-Gln143. As with peptide studies of this region (14Campbell A.P. Sheth H.B. Hodges R.S. Sykes B.D. Int. J. Pept. Protein Res. 1996; 48: 539-552Crossref PubMed Scopus (13) Google Scholar, 42Campbell A.P. Wong W.Y. Houston M. Schweizer F. Cachia P.J. Irvin R.T. Hindsgaul O. Hodges R.S. Sykes B.D. J. Mol. Biol. 1997; 267: 382-402Crossref PubMed Scopus (33) Google Scholar), a β-turn was found involving residues Asp134, Asn135, Lys136, and Tyr137. A second β-turn involving residues Pro139, Lys140, Thr141, and Cys142 could not be unambiguously determined, due mainly to the fact that resonances from residues Pro139, Lys140, and Thr141 could not be assigned due to NMR spectral overlap, resulting in poor definition of this region in the K122-4 pilin(29–150) structure. We expect that this portion of the receptor-binding domain does indeed form another β-turn by homology to the peptide studies and the crystal structures of PAK and N. gonorrhoeae strain MS-11. The first 5 residues of the receptor-binding domain of K122-4 pilin(29–150) is composed of residues in the last strand of the β-sheet resulting in a highly defined structured region that is not observed in isolated peptides from this C-terminal region (42Campbell A.P. Wong W.Y. Houston M. Schweizer F. Cachia P.J. Irvin R.T. Hindsgaul O. Hodges R.S. Sykes B.D. J. Mol. Biol. 1997; 267: 382-402Crossref PubMed Scopus (33) Google Scholar, 43Haas R. Schwarz H. Meyer T.F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9079-9083Crossref PubMed Scopus (93) Google Scholar). The K122-4 pilin(29–150) protein has an unusual charge distribution, as the charges are not distributed evenly on the surface of the molecule, but tend to be clustered in 5 regions (Fig.3 C). Basic residues are clustered in three regions: (i) Lys76, Lys80, Lys100, Lys109, and Lys111 (on the side of the pilus that faces the solvent), (ii) Arg30, Lys44, Lys136, and Lys140 (near the C-terminal binding loop), and (iii) Lys69 and Lys46 (on the C-terminal side of the helix near the acidic cluster). The acidic residues are clustered in two major regions: (iv) Asp49, Asp54, Glu68, Asp70, and Asp72 (along with G" @default.
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• W2021862499 date "2001-06-01" @default.
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• W2021862499 title "Structure of a Pilin Monomer fromPseudomonas aeruginosa" @default.
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• W2021862499 doi "https://doi.org/10.1074/jbc.m100659200" @default.
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