Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study

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 N-15-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 Ile(138)-Pro(139) 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 Asp(134)-Glu-Gln-Phe(137) and Pro(139)-Lys-Gly-Cys(142), 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., Sonnichsen, F. D., Kay, C. M., Hedges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hedges, 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 Pro(139)-Lys-Gly-Cys(142), but suggest a second beta-turn spanning Asp(132)-Gln-Asp-Glu(135). 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.