Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: Conformation of the bound peptide

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin prot ein strain PAK (residues 128-144) has been the target for the design of a v accine effective against P. aeruginosa infections. We have recently cloned and expressed a N-15-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (ci s) species in solution, arising from isomerization around a central Ile(138 )-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta -turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta -turn spanning Pro(139)-Lys-Gly-Cys(142). The cia isomer adopts on ly one well-defined type II beta -turn spanning Pro(139)-Lys-Gly-Cys(142) b ut displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(1 35). These turns have been implicated in cross-reactive antibody recognitio n. N-15-edited NMR spectroscopy was used to study the binding of the N-15-l abeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affini ty to the Fab, despite their different topologies; both isomers maintain th e conformational integrity of their beta -turns when bound; binding leads t o the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within reg ions of the trans and cia backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the f irst turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-sp ectrum vaccine capable of generating an antibody therapeutic effective agai nst the multiple strains of P. aeruginosa.