STRUCTURAL DYNAMICS OF H+/DRUG ANTIPORT IN AN ARCHAEAL MATE TRANSPORTER

Abstract

The multidrug and toxic compound extrusion (MATE) transporters are a ubiquitous family of efflux pumps that couples efflux of structurally and chemically diverse molecules to an inwardly-directed Na+ or H+ electrochemical gradient. MATE activity in bacteria contributes to antibiotic resistance, a serious public health threat. PfMATE is an archetype of the DinF subfamily of MATE transporters and the first H+-coupled MATE transporter to be structurally characterized. Despite multiple extracellular or outward facing (OF) structures with several ligands bound, the conformations stabilized by substrate and ion remained unclear. An intracellular facing (IF) conformation further confounded the identity of the coupling ion and the structural basis of alternating access. In this investigation, we integrated double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access in PfMATE. Spin label pairs monitoring the two sides of the transporter revealed helical rearrangements that are strictly dependent on a lipid environment and alter the orientation of a putative binding cavity in accordance with the tenets of alternating access. Acidic pH stabilized an IF conformation, whereas elevated pH (>7) and the substrate rhodamine 6G (R6G) stabilized an OF conformation. Protonation of a previously unidentified intracellular glutamate drives OF/IF isomerization of the transporter. Substitution of this glutamate compromised R6G resistance in cell growth assays. In addition, the presence of Na+ stabilized a unique intermediate at pH 4.0, which could be interpreted as an inward occluded conformation. Formation of this intermediate was found to be dependent on conserved residues in the N-lobe also shown to be critical to R6G resistance. Correlating PfMATE-mediated R6G binding and resistance with intrinsic (Trp) and extrinsic (spin labels) reporters of structural dynamics, we demonstrated that the functional cycle of drug efflux is facilitated by proton-dependent interconversion of the transporter between OF and IF conformations. Our findings define intermediate ligand-dependent conformational states of PfMATE, which can be framed into a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.