Structures of a Na
            <sup>+</sup>
            -coupled, substrate-bound MATE multidrug transporter

Lu, Min; Symerský, J.; Radchenko, Martha; Guo, Yi; Nie, Rongxin; Koide, Shohei

doi:10.1073/pnas.1219901110

Cited by 125 publications

(218 citation statements)

References 54 publications

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“…Coordinating glutamate and aspartate residues are a common feature of systems coupled to Na + (28,57,58). However, it is likely that some of these systems are actually H + selective, like the Na + -dependent ATP synthases; given that a large excess of Na + over H + is a feature of many natural environments, there is no evolutionary pressure to create binding sites that are highly Na + selective.…”

Section: Discussionmentioning

confidence: 99%

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Leone

Pogoryelov

Meier

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na + or H + , thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na + /H + selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H + selective, although not sufficiently to be functionally coupled to H + , owing to the large excess of Na + in physiological settings. The functional Na + specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H + selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na + -and H + -coupled systems in other families of membrane transporters and enzymes. molecular motor | ion-coupled transport | binding thermodynamics | energy transduction | membrane bioenergetics G radients in the electrochemical potential of H + or Na + across biological membranes sustain a wide range of essential cellular process. The resulting proton or sodium motive forces (pmf, smf) are the predominant energy source for secondary-active membrane transporters, which mediate the uptake of many substances required by the cell (1-3), and also enable pathogenic bacteria to protect themselves from human-made antibiotics and other toxic compounds (4-6). Downhill membrane permeation of Na + and H + across the membrane also powers the ATP synthase (7), which produces most of cellular ATP, and energizes the rotation of bacterial flagella (8). Thus, the importance of this mode of energy transduction in cells cannot be overstated. Nevertheless, little is known about the factors that control and modulate the specificity for Na + or H + in most of these processes.It seems clear, although, that there is no correlation between function type and ion specificity; that is, the same process in different species can be coupled to either Na + or H + (2, 5-7, 9-11). Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H + gradient despite the scarcity of H + and the potentially greater degree of H + leakage across the membrane at hig...

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Section: Discussionmentioning

confidence: 99%

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Leone

Pogoryelov

Meier

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Electron density for a Cs ϩ ion was observed in the x-ray structure of NorM_VC between Glu-255 and Asp-371; in NorM_NG, Glu-261 and Tyr-294 could coordinate a Na ϩ ion (8,9,33). Unlike both proteins mentioned above, NorM_PS is H ϩ dependent, and Glu-257 in NorM_PS is likely to stabilize the protein structurally as well as involved in DAPI binding.…”

Section: Asp-38 Is Involved In Hmentioning

confidence: 97%

“…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 drug binding pocket was published to be in the N-lobe cavity, which is halfway in the transmembrane region (9,11). Despite the report of the crystal structures of MATE transporters, no detailed biochemical characterization and substrate binding studies have been performed so far.…”

mentioning

confidence: 99%

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

Nie

Grell

Malviya

et al. 2016

Journal of Biological Chemistry

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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

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“…Generation of Mutants-Full-length NorM-NG was expressed with a decahistidine tag at the C terminus using a modified pET15b vector (7). Mutations were introduced into the norM-NG gene by using the QuikChange method (Agilent Technologies) and were confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Drug Resistance Assay-The drug export activities of NorM-NG variants were evaluated based on their ability to confer cellular resistance against cytotoxic chemicals (7,10). Drug susceptibility experiments were conducted in LB medium, with each assay repeated at least three times.…”

Section: Methodsmentioning

confidence: 99%

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

Radchenko¹,

Nie²,

2016

Journal of Biological Chemistry

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Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism.

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Structures of a Na ⁺ -coupled, substrate-bound MATE multidrug transporter

Cited by 125 publications

References 54 publications

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

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

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

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Structures of a Na + -coupled, substrate-bound MATE multidrug transporter

Cited by 125 publications

References 54 publications

On the principle of ion selectivity in Na + /H + -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

On the principle of ion selectivity in Na + /H + -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

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

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

Contact Info

Product

Resources

About

Structures of a Na ⁺ -coupled, substrate-bound MATE multidrug transporter

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor