Fig. 1: Molecular mechanisms for vesamicol and ACh recognition by VAChT.

a Schematic diagram of MBP-VAChT-DARPin. b Structure of vesamicol-bound VAChT in the lumen-facing conformation. c The cytosolic gate of VAChT. Two layers of gate residues from TMs 4, 5, 10, and 11 are shown as sticks on the side in magnified views. d Slice view of the vesamicol-bound VAChT structure. Vesamicol is shown in dark orange. TM1 and TM7 are shown as cartoon representations. e Left, the electrostatic surface potential of the vesamicol binding site calculated with the program APBS (http://www.poissonboltzmann.org); right, locations of the four acidic residues around vesamicol. f Details of the interaction between VAChT and vesamicol. The dashed lines represent hydrogen bonds, salt bridges, or electrostatic interactions (< 4.0 Å). The corresponding distances are indicated in angstroms. The residues involved in van der Waals and hydrophobic interactions (< 4.5 Å) are shown with side chains. g, h Binding of [³H]-vesamicol to different VAChT variants. One hundred percent binding was defined as the average [³H]-vesamicol signal bound to wild-type (WT) VAChT. The data are shown as the means ± SD; n = 3–10 biological replicates. i Structure of the ACh-bound VAChT. j Comparison of the structures of vesamicol- and ACh-bound VAChT. The root-mean-square deviation between these two structures is 0.9 Å. k Details of the interaction between VAChT and ACh. l Comparison of the ACh- and vesamicol (VES)-binding sites in VAChT. For clarity, only TM10 and TM11 are shown in pale green (vesamicol-bound) or light sky blue (ACh-bound). Asp398 and Tyr428 are shown as sticks. m Competitive binding analyses of ACh and [³H]-vesamicol with VAChT. One hundred percent binding was defined as the average [³H]-vesamicol signal bound to VAChT in the absence of ACh. The data are shown as the means ± SD; n = 3 biological replicates. n, o Comparison of the binding modes of ACh in VAChT and 5-HT in VMAT2 (n). The nonconserved residues within the substrate binding sites are shown in o.