The gram-negative bacterial pathogen Neisseria meningitidis is unusual in coding three homologues of the thiol-disulfide oxidoreductase, DsbA.1 Two are lipoproteins bound to the periplasmic side of the inner cell membrane, and the third is a soluble periplasmic protein. Like the prototypic DsbA from E. coli, the three homologues catalyze the post-translational formation of intramolecular disulfide bonds in substrate peptides to enable correct folding.2 However, their individual roles in cell function are not well understood. They are not critical for viability, but like other bacterial DsbAs, they contribute to the maturation and secretion of virulence factors.3
Structurally, the three homologues are similar despite pairwise sequence similarities ranging from 40-70%. All feature the characteristic DsbA hydrophobic groove, which is the binding site for substrate peptides prior to catalysis of oxidative folding.4 The groove is also hypothesized to bind the periplasmic loop of NmDsbB, the membrane protein that re-oxidizes the DsbA homologues to complete the catalytic cycle.5
Given its role in mediating the folding of virulence factors, DsbA is a potential target for novel antibacterial development. We have applied the techniques of Fragment-Based Drug Discovery,6 screening the three homologues of NmDsbA against our in-house fragment library by STD-NMR. Subsequently, we will validate the best STD hits by Surface Plasmon Resonance spectroscopy and by [1H,15N] HSQC. These validation steps will enable us to identify some fragments that bind to all three NmDsbA homologues, and others that bind to only one or two of the three. We will also evaluate binding sites based on X-ray crystal structures of NmDsbA1 and NmDsbA3, and the NMR solution structure of NmDsbA2, which we have solved in the course of this work.
Together, this information will assist us to design DsbA inhibitors with degrees of homologue specificity appropriate to the aims of our overall drug development strategy.