Cryo-EM reconstruction and functional characterization of VMAT2-tetrabenazine complex.

a, Predicted structural elements of VMAT2. The neurotransmitter substrate is bound at the central site (yellow, circle) between the two repeats comprised of TM1-6 and 7–12. The red and blue triangles depict the pseudo two-fold symmetric repeats. A disulfide bond (purple line) is predicted between lumenal loops LL1 and LL4. The N-linked glycosylation sites in LL1 are shown as red ‘Y’ shapes. b, Intrinsic fiducial strategy involves attachment of mVenus and GFP-Nb to the N- and C-terminus of VMAT2. c, Left panel, plots of [3H]-DTBZ saturation binding to wild type VMAT2 (black, circles) and chimera (red, squares). Symbols show the mean derived from n=3 technical replicates. Error bars show the s.e.m. Right panel, graphs of competition binding of 3H-DTBZ with unlabeled reserpine, error bars show the s.e.m. d, Plots of transport into vesicles using 1 and 10 µM 3H-serotonin for wild type VMAT2 (grey bars) and chimera (red bars). The bars show the means and points show the value for each technical replicate. Error bars show the s.e.m. e, f Occluded map of VMAT2-tetrabenazine complex (3.1 Å resolution, contour level 0.336). The mVenus and GFP-Nb fiducial is not shown for clarity.

Cryo-EM data collection, refinement and validation statistics.

VMAT2 conformation and residues involved in gating.

a, Overall view of the VMAT2-tetrabenazine (TBZ) complex. TBZ is shown in light green sticks with its map density in transparent surface. b, Closeup view of lumenal gating residues and TBZ, shown in stick representation together with transparent surface representation of their map density. c, Cytosolic gating residues, same representation as in b. d, Binding of DTBZ to various VMAT2 mutants in the lumenal and cytosolic gates including wild type (black), W318R (brown), W318H (light green), E127A (purple), W318F (orange), Y433A (forest green), F429A (blue), W318A (red) and F135A (teal). Data were normalized to wild type with error bars denoting standard deviation. e, Serotonin transport activity of lumenal and cytosolic gating residue mutants. Symbols show the mean derived from n=6 technical replicates with an identical color scheme to d. Asterisks denote statistical significance from wild type, with no significance being denoted with n.s. Data were normalized to wild type transport. Statistics were calculated in Graphpad Prism using a one-way ANOVA with Dunnett’s multiple comparison test. Error bars show the standard deviation. f, Alignment of five sequential residues of human VMAT2 (two residues on either side of W318) against their counterparts in Xenopus laevis (Xl), Helobdella robusta (Hr) and VMAT1, VAChT, and VPAT from humans. The residues which align with W318 in VMAT2 are shown in red.

Calculated Kd values of DTBZ for various single point mutants.

Polar networks.

a, Overall view showing three distinct polar networks. Polar residues involved in each network and TBZ are shown in sticks. b, Cartoon representation showing a zoomed view of polar network 1. c, Polar network 2. d, Polar network 3.

Tetrabenazine recognition and binding.

a, Chemical structure of tetrabenazine (TBZ). The blue dotted circle indicates the position of the hydroxyl group in DTBZ. b, Density associated with TBZ is shown in green transparent surface sharpened with a B-factor of −50 Å2. TBZ is shown in sticks fit to the density. c, Electrostatic potential of the TBZ binding site. d, e Binding site of TBZ, residues which are involved in binding are shown in sticks. TBZ is shown in light green sticks and the associated density. f, Plots of 3H-DTBZ saturation binding to wild type (black), V232L (pink), R189A (blue), E312Q (forest green), N34D (orange), N34T (light purple), N34A (teal), N34Q (brown) R189K (purple) L37F (red) and K138A (light green). g, Serotonin transport activity of mutants in TBZ binding site. Symbols show the mean derived from n=6 technical replicates with an identical color scheme to f. Asterisks denote statistical significance from wild type, with no significance being denoted with n.s. Statistics were calculated in Graphpad Prism using a one-way ANOVA with Dunnett’s multiple comparison test. Error bars show the s.e.m.

MD simulation systems of VMAT2 in the presence of TBZ, their properties and simulation durations.

pKa calculations performed by ProPKA 3.5.0.

Release of dopamine from the occluded binding site to the lumen through the lumen-facing vestibule and concurrent conformational changes in VMAT2.

Comparison of a, DA (yellow sticks) and b, TBZ (green sticks) binding to VMAT2, captured by MD simulations. Two water pathways are observed in the MD simulations of VMAT2 bound to DA (run DA_2 in Table 5). Water molecules and key residues are shown in sticks. c, Positions of dopamine (DA) (yellow van der Waals (VDW) spheres) with respect to hydrophobic gate composed of F135, W318, and F334 (purple VDW spheres), and charged residues lining the binding pocket at t = 0 ns, 30 ns, 100 ns, 170 ns, and 200ns. W318 side chain isomerization plays a critical role in mediating the opening/closure of the hydrophobic gate, accompanied by the reorientation of F135 side chain, permitting a flux of water molecules eventually giving rise to the destabilization and release of DA to the SV lumen by translocating through a hydrated channel. Waters within 10 Å radius from the center of mass (COM) of the hydrophobic gate residues are displayed.

MD simulations of VMAT2 in the presence of dopamine and the observed events

Mechanism of tetrabenazine inhibition, and the roles of gating residues and polar interactions networks.

a, Cartoon depicting substrate transport and tetrabenazine binding to VMAT2. Neurotransmitter (yellow cartoon) binds to the cytosolic open conformation before being released from the transporter in the lumenal open state. Tetrabenazine (green cartoon) binds to the lumenal-facing state and induces a conformational change to a high-affinity occluded conformation which is the resolved cryo-EM structure reported in this work. The VMAT2 – tetrabenazine complex highlighting significant features including both cytosolic (slashes) and lumenal gates (hexagon and pentagon depicting W318), the three polar networks (numbered circles) and relative location of the tetrabenazine binding site (green). b, Water penetrating through two pathways is involved in opening the lumenal gate. Dopamine is shown in yellow cartoon. c, Opening of W318 is associated with neurotransmitter release.