A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Fluman, Nir; Cohen-Karni, Devora; Weiss, Tali; Bibi, Eitan

doi:10.1074/jbc.m109.050658

Cited by 16 publications

(10 citation statements)

References 35 publications

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“…In reality, MdtM is a dynamic protein that undergoes conformational change during the drug efflux event 4 26 ; it is therefore probable that substrate interacts with other residues as it initially ‘explores’ the binding cavity of the protein 27 28 and subsequently translocates through the pore during the transport event. Such substrate-interacting residues have been identified in other MFS transporters 9 11 17 25 29 30 31 32 33 , and that information along with our MdtM model was used to guide identification of functionally related but not necessarily conserved residues in the latter 34 . This approach yielded an additional five residues (Q33, T120, M232, F261, and K342) as candidates for functioning in a substrate recognition/binding role in MdtM ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 97%

Insight into determinants of substrate binding and transport in a multidrug efflux protein

Alegre

Paul

Labarbuta

et al. 2016

Sci Rep

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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.The phenomenon of multidrug resistance -a serious global public health threat-arises principally from active efflux of drugs out of the cell cytoplasm by proteins that are integral membrane transporters. The 'transportome' of most bacteria contains numerous drug efflux proteins 1 , some of which are driven by energy released by ATP hydrolysis (the primary active transporters) and others by the energy stored in electrochemical gradients (the secondary active transporters).The majority of bacterial drug efflux proteins belong to the ubiquitous, large and diverse major facilitator superfamily (MFS) of secondary active transporters 2 . Although sequence similarity among the MFS is generally poor, all appear to follow the same structural template, their architecture consisting of either 12-or 14-transmembrane (TM) α -helices that are separated into six-or seven-helix bundle N-and C-terminal domains, respectively, related by a pseudo twofold symmetry and connected by a long cytoplasmic loop 3 . The N-and C-terminal halves of the transporter saddle a central substrate translocation pore, and both the N-and C-tails of the protein are located inside the cell. This structural arrangement implies that MFS proteins function via a single binding site, alternating access mechanism that is accompanied by a rocker switch-like movement of the two halves of the transporter, permitting access to the binding site(s) to swap between cytoplasm and periplasm 4 . The rocker switch mechanism supports at least three major conformational states during a dynamic transpor...

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Section: Resultsmentioning

confidence: 97%

Insight into determinants of substrate binding and transport in a multidrug efflux protein

Alegre

Paul

Labarbuta

et al. 2016

Sci Rep

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“…To this end, the affinity of purified, detergent-solubilized MdfA for its substrate tetraphenylphosphonium (TPP) was measured under various pH values, utilizing an intrinsic fluorescence assay that probes TPP-binding by its quenching effect on MdfA’s fluorescence (Fluman et al, 2009) (Figure S1A). Under increasing proton concentrations, the affinity for TPP was reduced (Figure 1A), indicating that high proton concentrations interfere with substrate binding.…”

Section: Resultsmentioning

confidence: 99%

“…TPP binding was measured as described (Fluman et al, 2009), with minor modifications described in Supplemental Experimental Procedures.…”

Section: Methodsmentioning

confidence: 99%

Dissection of Mechanistic Principles of a Secondary Multidrug Efflux Protein

et al. 2012

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SUMMARY Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/ proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multi-drug/proton antiport.

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“…In addition, the fluorescent cross-linking reagent must be a pump substrate. Fortunately, several such cross-linking compounds are available, such as MTS-rhodamine ( Loo and Clarke, 2002 ), BODIPY-FL-maleimide ( Husain et al, 2011 ), N -ethylmaleimide ( Loo and Clarke, 2002 ; Guan and Kaback, 2007 ), or monobromobimane ( Fluman et al, 2009 ). Thus, this approach could prove to be very useful for MOA studies for EPIs.…”

Section: Approaches To Study the Mechanism Of Action Of Epismentioning

confidence: 99%

Recent advances toward a molecular mechanism of efflux pump inhibition

2015

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Multidrug resistance (MDR) in Gram-negative pathogens, such as the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of Resistance-Nodulation-Division (RND)-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of RND-type efflux pump have been reported in the literature, and at least three of these EPI series were optimized in a pre-clinical development program. However, none of these compounds have been tested in the clinic. One of the major hurdles to the development of EPIs has been the lack of biochemical, computational, and structural methods that could be used to guide rational drug design. Here, we review recent reports that have advanced our understanding of the mechanism of action of several potent EPIs against RND-type pumps.

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A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Cited by 16 publications

References 35 publications

Insight into determinants of substrate binding and transport in a multidrug efflux protein

Insight into determinants of substrate binding and transport in a multidrug efflux protein

Dissection of Mechanistic Principles of a Secondary Multidrug Efflux Protein

Recent advances toward a molecular mechanism of efflux pump inhibition

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