Sequence and structural determinants of ligand-dependent alternating access of a MATE transporter

Abstract

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron–electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeonPyrococcus furiosus. Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux

“…Interestingly, a similar pattern was observed in the abovementioned studies of NorM-VC upon Na + recognition ( 34 ). While more detailed studies such as those carried out for NorM-VC and PfMATE ( 29 , 34 ) will be required to establish the relationship of Na + binding to transport, the evidence presented here strongly suggests that a Na + -binding site that modulates the transporter dynamics does exist in the N-lobe of VcmN.…”

“…These signals can be transformed into probability distributions for the distance between spin labels, P ( r ), with an associated confidence band ( 51 , 52 ). Through systematic analyses of signals from multiple spin-label pairs, studies of membrane transporters ( 53 , 54 , 55 , 56 , 57 , 58 , 59 ), including several MATEs ( 29 , 34 ), have examined how their conformational equilibria are modulated by ions and substrates. This modulation is manifested by shifts in the populations of the discrete distance components of P ( r ), reflecting changes in the conformational energy landscape of the protein ( 60 , 61 , 62 ).…”

“…NorM-VP and NorM-NG were the first MATEs reported to be driven by Na + ( 24 , 25 ), while more recently DinF-BH, NorM-PS, and VcmN have been described as H + -dependent ( 18 , 22 , 26 ). NorM-VC, by contrast, has been reported to be coupled to both Na + and H + ( 27 ); this dual specificity might also explain seemingly conflicting reports for ClbM ( 19 , 28 ) and PfMATE ( 17 , 23 , 29 ). Confusingly, monovalent cations such as K + , Rb + , and Li + have also been reported to influence substrate efflux in some cases ( 28 , 30 , 31 , 32 ).…”

“…This promiscuity of the N-lobe binding site might thus explain some of the reports of H + -dependent activity among MATEs. Nonetheless, additional binding sites for H + in the C-lobe or elsewhere in the structure have also been proposed as potential explanations for H + -coupling in this family ( 27 , 29 , 33 , 34 ).…”

DeerLab: A comprehensive toolbox for analyzing dipolar EPR spectroscopy data

“…Such titration or dose-response datasets have been analyzed using multi-Gaussian distribution models for the component distributions (Stein et al, 2015;Martens et al, 2016;Collauto et al, 2017;Barth et al, 2018;Jagessar et al, 2020). As discussed above, however, parameter-free distributions may often be a preferable choice.…”