Neisseria meningitidis and N. gonorrhoeae are causative agents of meningococcal disease and gonorrhoea respectively. These pathogens, particularly N. gonorrhoeae are recognised by the World Health Organization (WHO) as urgent threats to global health due to their increasing resistance to antimicrobials. New approaches are urgently needed to combat these pathogens.
Disulphide bonds are an important structural feature that proffers stability and function to many proteins. The process of disulphide bond catalysis is mediated by Dsb proteins in the periplasm of Gram-negative bacteria. A member of the Dsb family, DsbD is an enzyme essential for Neisserial viability rendering it an attractive target for antimicrobial development.
DsbD acts as an electron transport hub in the bacterial plasma membrane; transferring electrons sequentially and unidirectionally from the cytoplasm to target virulence substrates. Understanding the mechanism behind this unidirectional transfer is key to understanding Neisserial pathogenesis and ultimately to developing specific inhibitors. Our structure of the n-terminal domain of Neisseria DsbD revealed a flexible ‘Phen-cap loop’ postulated to control unidirectional electron flow (Smith et al., 2018).
In the present study, mutations have been introduced into key amino acids hypothesised to underpin the flexibility of the Phen-cap loop to determine their function. The structures of the mutant proteins were solved utilising X-ray crystallography to assess the effect of the mutations in the positioning of the Phen-cap loop. Overall, through structural and biochemical studies this work identified key amino acids that modulate the positioning of the Phen-cap loop, and ultimately regulate the reactivity of DsbD.
Further, the mechanistic understanding of DsbD has been utilised in a collaboration with Oracle Cloud Infrastructure to identify inhibitors in silico using cloud-based computing introducing a new method of inhibitor development.