Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Abstract

Multidrug and toxic compound extrusion (MATE) transportThe ability of all living organisms to protect themselves against toxic substances is essential for their survival. Organisms acquire this ability through evolutionary force (1). Due to the development of scientific knowledge, more and more medical drugs are being developed and applied. As a consequence, general defense mechanisms to resist antibiotics, drugs, and other toxic compounds have been evolved in bacteria (2). Being one of those defense mechanisms, the active extrusion of the toxic compounds from the cells plays an important role (3). In bacteria, the extrusion is often carried out by multidrug transporters, which are integral membrane proteins. They are classified into five distinct families: ATP-binding cassette, multidrug and toxic compound extrusion (MATE), 4 major facilitator superfamily, resistance nodulation division, and small multidrug resistance transporters (4).In 1999, the MATE family proteins were first recognized as a new group of the multidrug transporters, existing in all three domains of life (5). Most members of this family consist of a single chain of 450 -550 amino acid residues and exhibit 12 putative transmembrane helices (TMHs). Up to now, about 900 proteins have been annotated as MATE transporters on the basis of amino acid sequence similarities. Moreover, a subclassification of the MATE family has been suggested, NorM-, DinF-(DNA damage-inducible protein F), and eukaryotic subfamily (4, 6, 7). Various compounds can be recognized by NorM proteins, including dyes, fluoroquinolones, and aminoglycosides (7). As secondary active transporters, MATE proteins utilize transmembrane Na ϩ or H ϩ electrochemical gradients to extrude toxic compounds.So far atomic structures of four MATE transporters have been reported in their putative outward-facing states, in both drug-bound and/or drug-free states. NorM_VC from Vibrio cholerae and NorM_NG from Neisseria gonorrhoeae, are both Na ϩ driven, whereas PfMATE from Pyrococcus furiosus and DinF-BH from Bacillus halodurans are published to be H ϩ -dependent antiporters (8 -11). All these four proteins contain 12 TMHs with intracellular N and C termini, and possess a hydrophobic internal cavity, formed by two symmetric bundles with 6 TMHs each. The two bundles are linked by a cytoplasmic loop between the 6th and 7th TMH. All MATE proteins share about 40% sequence similarity, and they employ the same rockerswitch mechanism for substrate extrusion (12). In addition, the crystal structures have also revealed different locations of cationand substrate-binding sites, suggesting a mechanistic diversity among MATE transporters. In the structure of drug-bound NorM_NG, the binding site is located close to the membraneperiplasm interface, whereas in the structure of PfMATE, the

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

“…Previous investigations into residues involved in ion-coupling focused on the N-lobe cluster, such as D41, and to a lesser extent on residues in the C-lobe implicated in binding of Na + congeners in the crystal structures of NorM transporters 9,24,33,[39][40][41][42][43][44] . Our previously published work and the data reported here support the involvement of the N-lobe residues in PfMATE function as reflected in compromised drug resistance and reduced Trp quenching, which has been interpreted as impaired formation of a H + -dependent structural intermediate required for the transport cycle 26 .…”

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

“…We speculate that this competition might take place in different lobes for different substrates; PfMATE, for example, is known to recognize norfloxacin deep inside the N-lobe (13), causing conformational changes in TM1 (PDB ID code 3VVP) that would impair Na + (or H + ) binding to the site we have identified in this domain. However, biochemical evidence for, for example, NorM-PS, clearly indicates other substrates are directed to the C-lobe (21), where they would compete with H + . That distinct ion-recognition sites exist within each lobe might also suggest that the conformational mechanism of MATEs is unlike that of, for example, MFS transporters, whereby an N-and C-terminal domain rotate around a central site occupied by ions and/or substrates, for the most part preserving their internal structure (46).…”

“…NorM of Vibrio parahaemolyticus was the first MDR transporter shown to be driven by Na + (19), and other MATEs studied since then appear to share this property [e.g., NorM-NG (20) and ClbM (14)]. However, other prokaryotic MATEs have been reported to be coupled to H + instead [e.g., PfMATE (13), DinF-BH (12), and NorM-PS (21)] or, intriguingly, to both Na + and H + acting additively [e.g., NorM-VC (22)]. Other monovalent cations such as K + , Rb + , and Li + have also been reported to influence substrate efflux by some MATEs (23)(24)(25)(26).…”

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

“…Dual ion coupling has, for instance, recently been shown to occur in ClbM from Escherichia coli [76]. However, there may be crucial mechanistic differences in the ion coupling behavior of several NorM transporters (coupling to H + instead to Na + ), as previously proposed for NorM-PS from Pseudomonas stutzeri based on biochemical data [78]. The reasons for the different ion selectivity and the mechanistic diversity in the NorM family need to be clarified by further studies.…”