Conformational restriction shapes 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 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.

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

“…Previously, we proposed a mechanism for the AcrA efflux pump inhibitor (EPI) NSC 60339. 12 Using a combination of HDX-MS, MD simulations and cellular efflux assays, we suggested that NSC 60339 acts as a molecular wedge between the lipoyl and αβ barrel domains of AcrA, reducing its structural dynamics across all four domains. Our previous work utilised HDX-MS investigations at pH 6.0, reflecting the often more acidic conditions of the periplasmic environment, where AcrA resides.…”

“…Wild type, lipidated AcrA, was used for all functional assays and a previously established functional and soluble AcrA construct (AcrA s ), with its lipidation site removed by mutation, was used for structural and biophysical investigations as it provided the required sample homogeneity. 12,20 This delipidated AcrA construct was also utilized for MD simulations, with (de)protonation of the unique His285 residue used as a proxy for pH 7.4 and 6.0 conditions. His285 is located at the αβ barrel domain surface facing the lipoyl domain and is the only residue within AcrA that has a titratable protonation state change between mildly acidic and neutral regimes (with a pKa ∼6) ( Figure 1A ).…”

“…10 With homologs across other Gram-negative ESKAPE bacteria, enhanced mechanistic insight into this system can inform on wider RND behaviour as well as emerging efforts to inhibit the protein class to restore antibiotic efficacy. [11][12][13] The periplasmic environment AcrA resides in is distinct compared to the internal cytosolic and external environments of Gram-negative bacteria; it has a pH closer to the outside of the cell, concentrated cation populations, such as Mg 2+ (driven by the Donnan potential), and a peptidoglycan layer. 14 Critically, understanding collective contributions of protons and Mg 2+ becomes important when considering their putative fluctuations within the periplasm during the enteric journey of E. coli through a human gut (e.g.…”

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 (Fig.…”

Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins

“…The same complex is a target for inhibition to interfere with resistance development and restore antibiotic efficacy. MS analyses of this complex furnished molecular insights into multidrug adaptor protein function which could be useful for the development of new antimicrobial therapeutics [49].…”