Insights into the ion-coupling mechanism in the MATE transporter NorM-VC

Abstract

Bacteria have developed a variety of different mechanisms to defend themselves from compounds that are toxic to them, such as antibiotics. One of these defence mechanisms is the expulsion of drugs or other noxious compounds by multidrug efflux pumps. Multidrug and toxic compound extrusion (MATE) transporters are efflux pumps that extrude metabolic waste and a variety of antibiotics out of the cell, using an ion gradient as energy source. They function via an alternative-access mechanism. When ions bind in the outward facing conformation, a large conformational change to the inward facing conformation is induced, from which the ion is released and the extruded chemical compound is bound. NorM proteins, which are usually coupled to a Na + gradient, are members of the MATE family. However, for NorM-VC from Vibrio cholerae, it has been shown that this MATE transporter is additionally coupled to protons. How H + and Na + binding are coupled mechanistically to enable drug antiport is not well understood. In this study, we use molecular dynamics simulations to illuminate the sequence of ion binding event that enable efflux. Understanding this antiport mechanism is important to support the development of novel compounds that specifically inhibit the functional cycle of NorM transporters.3

Broadly conserved Na + -binding site in the N-lobe of prokaryotic multidrug MATE transporters

“…The fact is, however, that neither Rb + nor Cs + actually mimics Na + in any respect other than its charge. Indeed, a recent simulation study of outward-facing NorM-VC where the bound Rb + was replaced by Na + showed the ion to be stable only when an inward 400-mV electrostatic potential was applied across the protein (40). Evidently, that a membrane potential be present (chemical or electrostatic) is not a requirement for ion or substrate binding to secondary-active transporters (consider, for example, binding assays for purified, detergent-solubilized protein, or crystal structures).…”

On the ion coupling mechanism of the MATE transporter ClbM

“…It was shown recently that the N-lobe and the C-lobe ion binding sites from the sodium coupled MATE transporter NorM-VC [21,27] and NorM-NG [22] are solvent accessible, which is a prerequisite for ion binding and release, as e.g. discussed for the ion translocation mechanism of the membrane embedded domain from V-type ATPases [46], which is able to close the luminal water channel and thus prevents proton transport [47] to inactivate the enzyme.…”

“…Initially, the MATE transporter NorM from Vibrio cholerae (NorM-VC) was suggested to be coupled to sodium ions and protons [9], with the proton and sodium predicted to be harbored within the same binding site [27]. In contrast, recent measurements of ligand binding to NorM-VC indicated that the binding site residues solely coordinate protons [28]; however the authors of this study also claimed that a two-fold shift in the EC 50 (Na + ) for the E255Q mutant was observed, which indicates ion binding to E255 at some point during the conformational transformation.…”

How Ligand Binding Affects the Dynamical Transition Temperature in Proteins

“…Distinct interactions of these macromolecules with ligands and water molecules determine their structure and function, as in e. g. opening of ion‐channels upon binding of signaling molecules at the solvent accessible surface of the protein, or the folding of proteins and RNA, which is crucial for key activities such as enzyme catalysis. In addition, the recognition of ligands, including e. g. ions in soluble and membrane embedded proteins, small organic molecules, or even other macromolecules such as proteins, RNA and DNA can be governed by structural water molecules. Water access to a ligand binding site may be required in order for ligands to bind or exit; e. g. protons cannot be released from the V‐type ATPase if the protein channel is closed preventing water accessibility .…”