title: Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2.
abstract: 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 membrane-embedded 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.
authors.affiliation: Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Yaffe
authors.forename: Sebastian
authors.initials: S
authors.lastname: Radestock
authors.forename: Yonatan
authors.initials: Y
authors.lastname: Shuster
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D050493:Vesicular Monoamine Transport Proteins / Q000737:chemistry*
chemical_list: C493452:SLC18A2 protein, human; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins