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...

Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.

Background: Bacterial homologues of neurotransporters served as structural paradigms for interpretation of the functional data available for their eukaryotic counterparts. Results: We identified and characterized a close bacterial homologue of the rat vesicular monoamine transporter rVMAT2. Conclusion: BbMAT is a multidrug antiporter. Conserved membrane-embedded carboxyls play a role in substrate and proton transport. Significance: Understanding of the bacterial homologue should provide insights into rVMAT2.

of TM1A helix would not protrude into the membrane core. To further characterize the conformation of the TM1A helix, we used SMD simulations and pulled this helix relative to the scaffold or the core domain. The free energy profile was also endorsed our findings in the position of TM1A. These study indicates that changes in the environment can affect the equilibrium conformation of LeuT.