Solution Secondary Structure of a Bacterially Expressed Peptide from the Receptor Binding Domain ofPseudomonas aeruginosaPili 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.

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor

“…The phosphorylated SRR 2P peptide showed slightly higher NOE values for these central residues, suggesting a small reduction in mobility. More strikingly, the NOE values of residues throughout SRR-4ϕA 0P decreased substantially to those more characteristic of a random coil polypeptide (14,15). This indicates that the presence of the four aromatic sidechains dampens the backbone flexibility of the SRR peptides, possibly through the formation of dynamic hydrophobic clusters that lack any persistent secondary structure.…”

Dual-Function Vaccine for Pseudomonas aeruginosa : Characterization of Chimeric Exotoxin A-Pilin Protein

“…Type IV pili are composed of pilin polymers arranged in a helical structure with five subunits per turn (19,41). The portion of the pilin protein responsible for cell binding is located near the C terminus (amino acids 129 to 142) in a ␤-turn-␤-turn loop subtended from a disulfide bond (5,6,23,36). A 12-or 17-aminoacid sequence (depending on the specific strain) in this loop interacts with receptors on epithelial cells.…”

Functional Consequences of Sequence Variation in Bundlin, the Enteropathogenic Escherichia coli Type IV Pilin Protein

“…The relevant epitopes may not be recognized in the context of the intact pilus due to differences in three-dimensional conformation. For example, the solution structure of the isolated receptor binding peptide from the type IV pilin of P. aeruginosa is not identical to that of the intact pilin (9,20).…”