Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

Abstract

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a

Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD inSalmonellaTyphimurium

“…Correspondingly, AcrA and AcrE share a predicted secondary structure (Fig. 1a) 25 , which is also nearly identical to that of the experimentally determined structure of the AcrA from E.coli, with the exception of the divergent C-terminal region, which is predicted to be disordered and is not seen in the available cryo-EM structures 8, 9 . This allows creation of reliable homology models of AcrE, which we have previously reported 25 .…”

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from Salmonella

“…The action of the resistance-nodulation-division (RND) transporter AcrB provides the principal antimicrobial resistance efflux function in Salmonella enterica serovar Typhimurium (from here on, S. Typhimurium) [ 5 ]. In vivo AcrB forms a complex with the outer membrane factor (OMF) TolC and the periplasmic adaptor protein (PAP) AcrA, forming a functional tripartite complex with a 3:6:3 stoichiometry respectively, that spans both the inner and outer membrane [ 3 , 6 , 7 , 8 , 9 ]. Among the nine different tripartite efflux pumps involved in MDR in S .…”

Mutations in the TolC Periplasmic Domain Affect Substrate Specificity of the AcrAB-TolC Pump

“…AcrA is a multidomain periplasmic protein, with a hairpin-like topology, with both N- and C-terminal ends of its polypeptide chain contributing to each of the 4 principal domains, which are arranged in a string-like fashion. The membrane proximal (MPD) domain alongside the neighboring β-barrel and lipoyl domains are thought to interact with AcrB ( Du et al, 2014 ; McNeil et al, 2019 ), while an α-helical coiled-coil hairpin domain forms an extension, which in all models of assembly extends beyond AcrB further into the periplasm allowing AcrA to interact with TolC via an as-yet unconfirmed interface ( Mikolosko et al, 2006 ; Stegmeier et al, 2006 ; Lobedanz et al, 2007 ; Symmons et al, 2009 ; Du et al, 2014 ; Daury et al, 2016 ; Shi et al, 2019 ).…”

“…While the role of PAPs in substrate selection is less clear, several studies have highlighted their possible involvement in the case of metal-transporting RND-pumps ( De Angelis et al, 2010 ; Chacon et al, 2014 ) and substrate-dependent activation of both ABC- and RND-transporter based tripartite assemblies ( Tikhonova et al, 2007 ; Lin et al, 2009 ; Verchere et al, 2015 ). Adding to the complexity, there is also a notable redundancy amongst PAPs, which arises from their promiscuous interaction with a number of RND-partner pumps, allowing for added resilience and an ability to export similar, but different substrates ( Smith and Blair, 2014 ; McNeil et al, 2019 ).…”