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

Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2013; 110(6). DOI: 10.1073/pnas.1219901110
Source: PubMed


Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

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    • "Studies suggest that H+ coupling is operational in plants, which is substituted by Na+ in bacteria. Though structure and mechanism of action of MATEs have not been studied in detail, recent investigations revealed conformation and transport behaviour of MATE proteins34. Conversely to designated name “multidrug”, numerous studies revealed that MATE proteins have stringent substrate specificity and facilitate the movement of specific compounds5. "
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    ABSTRACT: Multidrug and Toxic compound Extrusion proteins (MATE) are a group of secondary active transporters with ubiquitous occurrences in all domains of life. This is a newly characterized transporter family with limited functional knowledge in plants. In this study, we functionally characterised two members of rice MATE gene family, OsMATE1 and OsMATE2 through expression in heterologous system, Arabidopsis. Expression of OsMATEs in Arabidopsis altered growth and morphology of transgenic plants. Genome-wide expression analysis revealed modulation of genes involved in plant growth, development and biotic stress in transgenic lines. Transgenic plants displayed sensitivity for biotic and abiotic stresses. Elevated pathogen susceptibility of transgenic lines was correlated with reduced expressions of defence related genes. Promoter and cellular localization studies suggest that both MATEs express in developing and reproductive organs and are plasma-membrane localised. Our results reveal that OsMATE1 and OsMATE2 regulate plant growth and development as well as negatively affect disease resistance.
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    • "Here, a selected antibody fragment mediated most of the crystal contacts and allowed its structure determination. Further successful examples mainly made use of antibody fragments derived from hybridoma technology [61,62] and recent examples used selected binding proteins based on proteins scaffolds such as DARPins [63] or fibronectin [64]. Camelid nanobodies become increasingly popular due to the recent successes in the field of GPCR structural biology [28,29]. "
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