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.
Highlights
Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat
Extensive biochemical studies of the prototypical Escherichia coli major facilitator superfamily (MFS) drug/H+ antiporter, MdfA, suggested the structural basis of substrate promiscuity lies in the presence of a large, flexible and complex substrate recognition cavity within the protein, which permits different substrates to interact with different regions of the cavity, and to form different interactions with it[7,8,9,10]
The crystal structure of EmrD, an E. coli MdfA homologue, captured in occluded conformation supports the notion of a large, hydrophobic, substrate-recognition cavity, the lack of any substrate molecule resolved in the structure precluded any detailed interpretation of a structural basis for substrate polyspecificity in this multidrug efflux representative of the MFS16
Summary
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. 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. Despite this much enhanced understanding, questions pertaining to multidrug recognition and transport by members of the MFS are still outstanding; for example, are the same residues involved in substrate recognition and binding in other conformations of the protein? are the same residues involved in recognition of all substrate types the protein is capable of transporting? We have attempted to shed light on these questions by performing a combination of in silico, biochemical, and mutagenesis studies on the E. coli MFS drug/H+ antiporter MdtM
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