title: Insight into determinants of substrate binding and transport in a multidrug efflux protein.
abstract: Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Kamela O
authors.initials: KO
authors.lastname: Alegre
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Paul
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Paola
authors.initials: P
authors.lastname: Labarbuta
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Law
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002701:Chloramphenicol / Q000737:chemistry; D003198:Computer Simulation; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009861:Onium Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D017920:Antiporters; D029968:Escherichia coli Proteins; C574623:MdtM protein, E coli; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D002701:Chloramphenicol; C013289:tetraphenylphosphonium