title: Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.
abstract: The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.
authors.affiliation: Protein Engineering Network of Centres of Excellence (PENCE), 713 Heritage Medical Research Building, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
authors.forename: A P
authors.initials: AP
authors.lastname: Campbell
authors.forename: D L
authors.initials: DL
authors.lastname: Bautista
authors.forename: B
authors.initials: B
authors.lastname: Tripet
authors.forename: W Y
authors.initials: WY
authors.lastname: Wong
authors.forename: R T
authors.initials: RT
authors.lastname: Irvin
authors.forename: R S
authors.initials: RS
authors.lastname: Hodges
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D001428:Bacterial Vaccines; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D036781:Fimbriae Proteins; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry*; D018933:Pili, Sex; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics; D014614:Vaccines, Synthetic
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012996:Solutions; D014614:Vaccines, Synthetic; D036781:Fimbriae Proteins