Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Tirosh, Osnat; Sigal, Nadejda; Gelman, Amir; Sahar, Nadav; Fluman, Nir; Siemion, Shira; Bibi, Eitan

doi:10.1073/pnas.1203632109

Cited by 35 publications

(48 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A dual role for carboxyl residues was demonstrated in EmrE, where a conserved Glu (E14) can alternately bind either the substrate or proton and thus couple the two processes (29,30). Similar mechanisms also were proposed in the transport cycle of other MFS transporters such as LmrP (31) and MdfA (32). Interestingly, when the position equivalent to E313 in the close homolog VAChT was replaced by Asp or Gln, the mutant protein was still able to transport acetylcholine (33).…”

Section: Discussionmentioning

confidence: 78%

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Yaffe

Radestock

Shuster

et al. 2013

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

View full text Add to dashboard Cite

Vesicular monoamine transporter 2 (VMAT2) catalyzes transport of monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Here we present a homology model of VMAT2, which has the standard MFS fold, that is, with two domains of six transmembrane helices each which are related by twofold pseudosymmetry and whose axis runs normal to the membrane and between the two halves. Demonstration of the essential role of a membraneembedded glutamate and confirmation of the existence of a hydrogen bond probably involved in proton transport provide experimental evidence that validates some of the predictions inherent to the model. Moreover, we show the essential role of residues at two anchor points between the two bundles. These residues appear to function as molecular hinge points about which the two six transmembrane-helix bundles flex and straighten to open and close the pathways on either side of the membrane as required for transport. Polar residues that create a hydrogen bond cluster form one of the anchor points of VMAT2. The other results from hydrophobic interactions. Residues at the anchor points are strongly conserved in other MFS transporters in one way or another, suggesting that interactions at these locations will be critical in most, if not all, MFS transporters.ion coupling | multidrug resistance | membrane proteins | neurotransmitter transporter | homology modeling T ransport and storage of neurotransmitters in synaptic vesicles allow their regulated release from the presynaptic cell into the synaptic cleft. The neurotransmitter molecules are accumulated in synaptic vesicles by vesicular neurotransmitter transporters (1-3). Transport of monoamines (serotonin, dopamine, histamine, adrenaline, and noradrenaline) is carried out by the vesicular monoamine transporter (VMAT) family, which includes two isoforms, VMAT1 and VMAT2, in a process that involves the exchange of two protons for one substrate molecule (1-3). The proton electrochemical gradient necessary for transport is generated by the vesicular H + -ATPase (V-ATPase). The structural basis for the function of VMAT remains unknown. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Most MFS transporters contain 12 transmembrane (TM) helices, and crystal structures revealed that the 12 TM helices are arranged in two domains of six TMs each, which are related by a twofold pseudosymmetry with an axis that runs normal to the membrane and between the two halves (4-9). Furthermore, analysis of the lactose permease (LacY) crystal structure revealed the presence of inverted topology repeat units within each of the domains (10). That is, the first three helices of each domain are structurally related to the second three helices of that domain by a twofold pseudosymmetry ax...

show abstract

Section: Discussionmentioning

confidence: 78%

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Yaffe

Radestock

Shuster

et al. 2013

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

View full text Add to dashboard Cite

show abstract

“…V335 TM10.5 E is able to re-establish cationic drug resistance of the inactive mutant E26T [35]. Mutant variants L119 TM4.3 E, V231 TM7.4 E, G354 TM11.4 E and M358 TM11.3 E are able to establish divalent cationic drug resistance in MdfA, which otherwise lacks such capability due to the exchange of only one proton per transport cycle [36]. In the C In structure of MdfA, all residues described above except for V231 face the central cavity.…”

Section: Multidrug Resistancementioning

confidence: 99%

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

et al. 2015

View full text Add to dashboard Cite

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.

show abstract

“…As shown recently, the stoichiometry of one proton per drug renders MdfA inactive in efflux of divalent cationic (dicationic) drugs, which require import of two protons per transport cycle 8 . The underlying reason is that such an antiport reaction (export of drug 2 þ in exchange for a single H þ ) involves net charge efflux that opposes the membrane potential (Dc, interior negative).…”

mentioning

confidence: 99%

“…Recent progress has shed light on the mechanism of proton transport by MdfA, in which a membrane-embedded acidic residue facilitates proton binding and transport 4 . Mutagenesis experiments indicate that increasing the proton stoichiometry of MdfA from 1 to 2 is possible by introducing an additional acidic residue in a number of membrane-embedded locations 8 . LmrP, which works with a stoichiometry of up to three protons, contains three membraneembedded acidic residues; each functions as a module that facilitates the transport of a single proton 7 .…”

mentioning

confidence: 99%

Export of a single drug molecule in two transport cycles by a multidrug efflux pump

et al. 2014

Self Cite

View full text Add to dashboard Cite

Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of divalent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of divalent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.

show abstract

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Cited by 35 publications

References 27 publications

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

Export of a single drug molecule in two transport cycles by a multidrug efflux pump

Contact Info

Product

Resources

About