Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ₁: S258-T295; JlpAΔ₂: Q332-A354) and assessed the Hsp90α-binding affinity kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residue Q332-A354 is of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ₂ to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.