Sequence and Structural Determinants of Ligand-dependent Alternating Access of a MATE Transporter

Abstract

MATE transporters are ubiquitous ion-coupled antiporters that extrude structurally-and chemically-dissimilar molecules and have been implicated in conferring multidrug resistance.Here, we integrate Double Electron Electron Resonance (DEER) in conjunction with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of liganddependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus 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 inwardfacing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. PfMATE isomerization between outward-facing and inwardfacing conformations 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.

Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1

“…Therefore, we explored catalysis and stability of mG6PC1 when purified into LMNG micelles supplemented with the model lipid bilayer species 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC) or cholesteryl hemisuccinate (CHS), a cholesterol analog with higher aqueous solubility (50). While POPC and CHS are typical lipid and sterol representatives in biophysical and structural studies (51)(52)(53)(54)(55)(56), POPC has been used specifically as an ER-mimicking lipid (57).…”

DeerLab: a comprehensive software package for analyzing dipolar electron paramagnetic resonance spectroscopy data

“…Either the mole fractions or the underlying equilibrium constant can be included among the fitting parameters. 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, non-parametric distributions may often be a preferable choice.…”

On the ion coupling mechanism of the MATE transporter ClbM

“…The proposed Na + binding site [24] also cannot explain the fact that this MATE transporter [14] and VcmN from Vibrio cholerae [17] have been crystallized in bent and straight conformations under different pH conditions, indicating a protonation driven conformational change in the outward-open state. After protonation of D41 (PfMATE) [14] or D35 (VcmN) [17] in the outward-open state, the straight conformation is transformed to the bent conformation, as also supported by fluorescence [25] and DEER spectroscopy [26]. In disagreement with this, another recent crystallographic study claimed that the conformational change may not be pH dependent, but Na + coupled [19] and thus additional Na + transport assays are needed to clarify the ion coupling preference.…”

“…Accessibility pathways for Na + [21,22] and H + [23] transport to the essential glutamate have previously been described in detail for the outward-facing conformation. However, a recent study questioned the H + selectivity for PfMATE and proposed the existence of a highly conserved Na + binding site in the MATE family [24]; this idea is, however, in disagreement with structural data [14] as well as recent fluorescence [25] and Double Electron Electron Resonance (DEER) [26] measurements. Transport of Na + during its catalytic cycle has not yet been demonstrated experimentally for PfMATE, while protons cause the conformational change from the outward-facing to the inward-facing conformation, as shown by DEER measurements [26].…”

“…However, a recent study questioned the H + selectivity for PfMATE and proposed the existence of a highly conserved Na + binding site in the MATE family [24]; this idea is, however, in disagreement with structural data [14] as well as recent fluorescence [25] and Double Electron Electron Resonance (DEER) [26] measurements. Transport of Na + during its catalytic cycle has not yet been demonstrated experimentally for PfMATE, while protons cause the conformational change from the outward-facing to the inward-facing conformation, as shown by DEER measurements [26]. The proposed Na + binding site [24] also cannot explain the fact that this MATE transporter [14] and VcmN from Vibrio cholerae [17] have been crystallized in bent and straight conformations under different pH conditions, indicating a protonation driven conformational change in the outward-open state.…”