Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Abstract

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

Molecular insights into the determinants of substrate specificity and efflux inhibition of the RND efflux pumps AcrB and AdeB

“…A combination of computational (ensemble docking) and experimental approaches ( in vitro AcrA binding studies using surface plasmon resonance, as well as in vivo limited proteolysis) suggested that both compounds target AcrA, the MFP/PAP component of the tripartite AcrAB-TolC complex. NSC60339 is thought to bind to the hinge region (linking α-hairpin- and lipoyl-domains) of AcrA, thereby constricting conformational flexibility and reducing its ability to transduce signal to AcrB possibly inhibiting functional rotation and efflux [182]. NSC33353, however, is suggested to bind the AcrA membrane proximal (MP) domain (), presumably interfering with functional interactions between AcrA and AcrB.…”

Understanding the biofilm development of Acinetobacter baumannii and novel strategies to combat infection

“…These scientists used a "molecular wedge" technique, primarily a compound known as NSC 60339, that could successfully disrupt the function of bacterial efflux pumps. The resulting disruption prevents bacteria from releasing antibiotics, increasing the efficacy of these antimicrobial agents against bacterial infections [133].…”

Exploring the inhibitory potential of in silico-designed small peptides on Helicobacter pylori Hp0231 (DsbK), a periplasmic oxidoreductase involved in disulfide bond formation

“…Periplasmic proteins are convenient targets for the development of new antivirulence agents or antibiotic adjuvants against Gram-negative bacteria ( Pandeya et al, 2020 ). The recently exploited periplasmic targets include, e.g., AcrA—acriflavine resistance protein A (its inactivation results in increased sensitivity to many antimicrobials) ( Russel Lewis et al, 2023 ), MtrD—resistance-nodulation-cell division (RND) family transporter protein (its inhibition impairs the function of the multidrug efflux pomp) ( Lyu et al, 2022 ), SurA—periplasmic chaperone (its inhibition results in increased antibiotic susceptibility and decreased virulence) ( Bell et al, 2018 ), LptA—lipopolysaccharide transport protein (its inhibition alters or blocs transport of lipopolysaccharides) ( Sinha et al, 2022 ), and PelA—deacylase (its inhibition leads to disruption of exopolysaccharide production and biofilm formation) ( Razvi et al, 2023 ).…”