Fragment-Based Studies of Three DsbA Homologues from <em>Neisseria meningitidis</em>: Implications of Ligand Specificity for Design of Novel Antivirulence Agents — ASN Events
  1. Schedule
  2. Session 12: Poster Session D - Including Happy Hour & Trade Display
  3. Fragment-Based Studies of Three DsbA Homologues from Neisseria meningitidis: Implications of Ligand Specificity for Design of Novel Antivirulence Agents

Fragment-Based Studies of Three DsbA Homologues from Neisseria meningitidis: Implications of Ligand Specificity for Design of Novel Antivirulence Agents (#411)

Martin L Williams 1 2 , Biswaranjan Mohanty 1 2 , Martin J Scanlon 1 2
  1. Monash University, Parkville, VIC, Australia
  2. ARC Centre of Excellence for Coherent X-ray Science, Melbourne

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 inner cell membrane; the third is a soluble periplasmic protein. Like the prototypic DsbA from E. coli, they 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 very similar despite pairwise sequence similarities ranging from 40-70%. All feature the characteristic DsbA hydrophobic groove, the binding site for substrate peptides prior to catalysis of oxidative folding.4 The groove also binds 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 correct 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 homologues of NmDsbA against our in-house fragment library by STD-NMR. Subsequently, we validated the best STD hits by Surface Plasmon Resonance spectroscopy and by [15N,1H]-HSQC. These validation steps have enabled 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 are also able to 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 as appropriate to the aims of our overall drug development strategy.

1. Tinsley, C. R. et al (2004). Three Homologues, Including Two Membrane-bound Proteins, of the Disulfide Oxidoreductase DsbA in Neisseria meningitidis. Journal of Biological Chemistry 279:27078-27087.
2. Kadokura, H. & Beckwith, J. (2010). Mechanisms of Oxidative Protein Folding in the Bacterial Cell Envelope. Antioxidants & Redox Signaling 13:1231-1246.
3. Heras, B. et al (2009). DSB proteins and bacterial pathogenicity. Nat Rev Microbiology 7:215-225.
4. Paxman, J. J. et al (2009). The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes. Journal of Biological Chemistry 284:17835-17845.
5. Inaba, K. et al (2006). Crystal Structure of the DsbB-DsbA Complex Reveals a Mechanism of Disulfide Bond Generation. Cell 127:789-801.
6. Murray, C. W. et al (2012). Experiences in fragment-based drug discovery. Trends in Pharmacological Sciences 33:224-232