Structural mechanisms for VMAT2 inhibition by tetrabenazine
- Department of Structural Biology, University of Pittsburgh, United States
- Laufer Center for Physical and Quantitative Biology, and Department of Biochemistry and Cell Biology, School of Medicine, Stony Brook University, United States
Figures
Figure 1 with 3 supplements
Cryo-electron microscopy (cryo-EM) reconstruction and functional characterization of vesicular monoamine transporter 2 (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 transmembrane (TM)1–6 and 7–12. The red …
Figure 1—figure supplement 1
Biochemical characterization, construct design, and sequence conservation of vesicular monoamine transporter 2 (VMAT2).
(a) The mVenus and GFP-Nb was fused to the N- and C-terminus of VMAT2 and the length of the termini is varied to find constructs which can be studied by cryo-electron microscopy (cryo-EM) and retain …
Figure 1—figure supplement 2
Cryo-electron microscopy (cryo-EM) data processing of the vesicular monoamine transporter 2 (VMAT2)-tetrabenazine complex.
A representative micrograph (defocus –1.3 µm) is shown (scale bar equals 80 nm). The workflow depicts the data processing scheme used to reconstruct VMAT2. Two datasets were collected comprising …
Figure 1—figure supplement 3
Cryo-electron microscopy (Cryo-EM) maps and interpretation of vesicular monoamine transporter 2 (VMAT2) reconstruction.
(a) Cryo-EM density colored by local resolution estimation. (b) FSC curves for cross-validation, the unmasked map (blue), loose mask (green), tight mask (red), and the reported corrected (purple) …
Figure 2 with 1 supplement
Vesicular monoamine transporter 2 (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 …
Figure 2—figure supplement 1
Sequence alignment of vesicular monoamine transporter 1 (VMAT1), VMAT2, VAChT, and VPAT.
The positions of mutated residues are shown in red boxes. The human variants are shown in blue boxes (Yariv et al., 2023; Pei et al., 2008).
Figure 3
Polar networks.
(a) Overall view showing three distinct polar networks. Polar residues involved in each network and tetrabenazine (TBZ) are shown in sticks. (b) Cartoon representation showing a zoomed view of polar …
Figure 4 with 6 supplements
Tetrabenazine (TBZ) recognition and binding.
(a) Chemical structure of TBZ. The blue dotted circle indicates the position of the hydroxyl group in dihydrotetrabenazine (DTBZ). (b) Density associated with TBZ is shown in green transparent …
Figure 4—figure supplement 1
Point mutants in tetrabenazine (TBZ) binding site.
(a) 2D cartoon of the TBZ binding site showing only highlighted residues. Green, red, and blue indicate hydrophobic, negatively, or positively charged properties of the side chains (Schrödinger, 2023…
Figure 4—figure supplement 2
Tetrabenazine (TBZ) docking and molecular dynamics (MD) simulations.
(a–c) Time evolution of (a) root-mean-square-deviations (RMSDs) in vesicular monoamine transporter 2 (VMAT2) Cα-coordinates from those resolved in the cryo-electron microscopy (cryo-EM) structure, (b…
Figure 4—figure supplement 3
Effects of protonation states of tetrabenazine (TBZ) and selected vesicular monoamine transporter 2 (VMAT2) residues on the time evolution of TBZ (heavy atoms) and VMAT2 (α-carbons) root-mean-square-deviations (RMSDs) from their original (cryo-electron microscopy [cryo-EM]-resolved) positions.
Results are presented for three different types of simulations (TBZ_2–TBZ-4; see Table 3). In each case, the top panels (a), (b), and (c) display the RMSDs in the heavy atoms of TBZ and the lower …
Figure 4—video 1
Binding and coordination of neutral tetrabenazine (TBZ) (cyan van der Waals [vdW] representation) observed in a 180 ns molecular dynamics (MD) simulation (run 1).
Three runs were performed for vesicular monoamine transporter 2 (VMAT2) embedded in a lipid bilayer in the presence of TBZ, where E312 and D399 were protonated. We focus here on the time evolution …
Figure 4—video 2
Binding and coordination of neutral tetrabenazine (TBZ) (cyan van der Waals [vdW] representation) observed in a 180 ns molecular dynamics (MD) simulation (TBZ_1 run 2), in the same format as Figure 4—video 1.
TBZ slightly altered its position within the same binding pocket, to stabilize the pose illustrated in Figure 4—figure supplement 2f.
Figure 4—video 3
Binding and coordination of neutral tetrabenazine (TBZ) (cyan van der Waals [vdW] format) observed in a 180 ns molecular dynamics (MD) simulation (TBZ_1 run 3), in the same format as Figure 4—video 1.
TBZ adopts a pose similar to that resolved in the cryo-electron microscopy (cryo-EM) structure.
Figure 5 with 2 supplements
Release of dopamine (DA) from the occluded binding site to the lumen through the lumen-facing vestibule and concurrent conformational changes in vesicular monoamine transporter 2 (VMAT2).
Comparison of (a) DA (yellow sticks) and (b) tetrabenazine (TBZ) (green sticks) binding to VMAT2, captured by molecular dynamics (MD) simulations. Two water pathways are observed in the MD …
Figure 5—figure supplement 1
Comparisons of binding poses of substrate (dopamine [DA] and serotonin) and inhibitor (tetrabenazine [TBZ]) from docking simulations and molecular dynamics (MD) simulations under different protonation states of substrate-coordinating and/or gating residues.
(a) Docking of DA and (b) serotonin to the cryo-electron microscopy (cryo-EM)-resolved vesicular monoamine transporter 2 (VMAT2). The most energetically favorable poses are shown for both …
Figure 5—video 1
Release of dopamine (DA) to the vesicular lumen (yellow van der Waals [VDW] spheres), observed after protonating D33, E312, D399, and D426 (red vdW spheres). Related to Figure 5.
The video represents a 200 ns molecular dynamics (MD) simulation of system DA_4 in Table 5.
Figure 6 with 2 supplements
Mechanism of tetrabenazine inhibition, and the roles of gating residues and polar interactions networks.
(a) Cartoon depicting substrate transport and tetrabenazine binding to vesicular monoamine transporter 2 (VMAT2). Neurotransmitter (yellow cartoon) binds to the cytosolic-open conformation before …
Figure 6—figure supplement 1
Comparison of the vesicular monoamine transporter 2 (VMAT2)-tetrabenazine (TBZ) structure with the predicted AlphaFold structure and with other major facilitator superfamily (MFS) transporters.
(a) Comparison of the cryo-electron microscopy (cryo-EM) structure (light blue) vs. AlphaFold (navy). The position of TM1, -2, -4, -7, -8, -10, and -11 and LL4 in the cryo-EM structure shows the …
Figure 6—figure supplement 2
Human variants of vesicular monoamine transporter 2 (VMAT2).
P316A, P237H, and P387L localize to LL4 and the lumenal ends of TM5 and TM9, respectively.
Videos
Video 1
Same as Figure 4—videos 1–3, with neutral tetrabenazine (TBZ), but with the additional protonation of D426 (TBZ_3 run 1).(Related to Table 3).
TBZ adopted the predominant pose similar to that resolved in the cryo-electron microscopy (cryo-EM) structure.
Video 2
Molecular dynamics (MD) simulation of the binding and coordination of protonated tetrabenazine (TBZ) (TBZ+; pink van der Waals [vdW] format) to vesicular monoamine transporter 2 (VMAT2) with protonated E312 and D399.
(Related to Table 3). This is a 100 ns run, termed TBZ_2 run 1. The same format as Figure 4—videos 1–3 is adopted for the coordinating residues. TBZ tends to alter its binding pose to approximate …
Video 3
Same as Video 1, except for the protonation of tetrabenazine (TBZ) (TBZ+ shown in pink van der Waals [vdW] representation).
(Related to Table 3).This is a 100 ns run, termed TBZ_4 run 1. In the presence of protonation, TBZ preferentially samples the pose observed in Figure 4—video 2, Video 2.
Video 4
Fluctuations in dopamine binding pose (DA; yellow van der Waals [vdW] spheres) within its binding pocket, observed in a 100 ns molecular dynamics (MD) simulation of vesicular monoamine transporter 2 (VMAT2) in the presence of DA (system DA_2 in Table 5). Related to Table 5.
VMAT2 E312 and D399 are protonated; and D33 and D426 are deprotonated. Hydrophobic gate residues F135, W318, and F334 are displayed in purple vdW spheres, and acidic and basic residues K138 and R189 …
Tables
Table 1
Cryo-electron microscopy (cryo-EM) data collection, refinement, and validation statistics.
| VMAT2-TBZ(EMDB-41269)(PDB 8THR) | |
|---|---|
| Data collection and processing | |
| Magnification | 77,279 |
| Voltage (kV) | 300 |
| Electron exposure (e–/Å2) | 60 |
| Defocus range (μm) | –1.0 to –2.5 |
| Pixel size (Å) | 0.647 |
| Symmetry imposed | C1 |
| Initial particle images (no.) | ~10,000,000 |
| Final particle images (no.) | 65516 |
| Map resolution (Å) FSC threshold | 3.12 0.143 |
| Map resolution range (Å)* | 6.6–2.8 |
| Refinement | |
| Initial model used (PDB code) | |
| Model resolution (Å) FSC threshold | 3.7 0.5 |
| Model resolution range (Å) | 20.4–3.7 |
| Map sharpening B-factor Å2 | |
| Model composition Non-hydrogen atoms Protein residues Ligands | 2937 389 1 |
| B-factors Å2 Protein Ligand | 28.99 55.22 |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.0004 (0) 0.37 (15) |
| Validation MolProbity score Clashscore Poor rotamers (%) | 1.23 4.34 0.96 |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 98.0 2.00 0.00 |
-
*
Local resolution range at 0.5 FSC.
Table 2
Calculated Kd values of dihydrotetrabenazine (DTBZ) for various single-point mutants.
| Mutant | Kd (nM) |
|---|---|
| WT | 15±3 |
| N34A | ND |
| N34D | ND |
| N34Q | ND |
| N34T | ND |
| E127A | 15±6 |
| F135A | ND |
| R189A | ND |
| V232L | ND |
| E312Q | ND |
| W318A | ND |
| W318F | 24±16 |
| W318H | ND |
| W318R | ND |
| F429A | 7.7±0.6 |
| Y433A | ND |
Table 3
Molecular dynamics (MD) simulation systems of vesicular monoamine transporter 2 (VMAT2) in the presence of tetrabenazine (TBZ), their properties and simulation durations.
| System ID | Protonated residues | Bound ligand | Duration (ns) | Waters in binding pocket* | RMSD in VMAT2 Cα-atoms (Å)† | RMSD in TBZ heavy atoms (Å) ‡ |
|---|---|---|---|---|---|---|
| TBZ_1 | E312 and D399 | TBZ | 3×180 | 2.8±1.3 | 2.4±0.3 | 2.7±0.5 |
| TBZ_2 | TBZ+ | 2×100 | 8.1±1.4 | 2.4±0.4 | 5.9±1.4 | |
| TBZ_3 | E312, D399, and D426 | TBZ | 3×100 | 4.6±1.4 | 2.7±0.5 | 3.5±1.3 |
| TBZ_4 | TBZ+ | 2×100 | 14.3±2.4 | 2.3±0.2 | 3.7±0.4 |
-
*
Number of water molecules within 3.5 Å of TBZ (or TBZ+) averaged over multiple MD snapshots.
-
†
RMSDs (root-mean-square-deviations) of VMAT2 Cα-atoms from their initial (cryo-EM resolved) positions.
-
‡
RMSD of the heavy atoms of TBZ from their cryo-EM-resolved positions. All averages and standard deviations were calculated between 50 and 100 ns portion of the MD trajectories.
Table 4
pKa calculations performed by PROPKA 3.5.0.
| Residue | pKa with TBZ | pKa without TBZ | Model pKa |
|---|---|---|---|
| D33 | 5.48 | 5.43 | 3.8 |
| D121 | 4.46 | 4.46 | 3.8 |
| D123 | 4.75 | 4.75 | 3.8 |
| D213 | 3.23 | 3.23 | 3.8 |
| D214 | 4.95 | 4.95 | 3.8 |
| D262 | 6.44 | 6.44 | 3.8 |
| D291 | 4.44 | 4.44 | 3.8 |
| D399 | 7.64 | 7.56 | 3.8 |
| D411 | 2.74 | 2.74 | 3.8 |
| D426 | 6.38 | 6.35 | 3.8 |
| D460 | 8.19 | 8.07 | 3.8 |
| E120 | 3.06 | 3.06 | 4.5 |
| E127 | 4.77 | 4.77 | 4.5 |
| E215 | 4.27 | 4.27 | 4.5 |
| E216 | 3.91 | 3.91 | 4.5 |
| E244 | 4.52 | 4.52 | 4.5 |
| E278 | 4.22 | 4.22 | 4.5 |
| E312 | 6.71 | 7.46 | 4.5 |
| E321 | 4.53 | 4.53 | 4.5 |
| K138 | 9.86 | 10.04 | 10.5 |
| R189 | 9.10 | 10.02 | 12.5 |
| TBZ | 8.57 | 10 |
-
The cryo-EM structure (with or without TBZ) was used for pKa calculations using PROPKA 3.5.0. Key residues of interest are written in boldface.
Table 5
Molecular dynamics (MD) simulations of vesicular monoamine transporter 2 (VMAT2) in the presence of dopamine and the observed events.
| System # | Protonated residues | Duration(ns) | Waters in binding pocket* | VMAT2 RMSD† fromcryo-EM (Å) | Observed events |
|---|---|---|---|---|---|
| DA_0 | None | 2×100 | 7.7±1.5 | 2.6±0.2 | Salt bridge formation between DA and D399; influx of water into the binding pocket. |
| DA_1 | D399 | 2×100 | 10.5±2.2 | 2.2±0.2 | Salt bridges formation between DA and E312; influx of water into the binding pocket. |
| DA_2 | E312, D399 | 2×100 | 8.8±2.1 | 2.2±0.3 | Fluctuations in DA binding pose while remaining within the binding pocket. Formation of two water wires. |
| DA_3 | E312, D399, D426 | 2×100 | 8.8±2.1 | 2.5±0.2 | Fluctuations in DA binding pose; dislocation of DA from its binding site in one of the two simulations. |
| DA_4 | E312, D399, D426, D33 | 1×100 1×200 | 11.7±2.3 | 2.6±0.3 | Fluctuations in DA binding pose; dislocation and release of DA in the 200 ns run. |
-
*
Number of water molecules within 3.5 Å of dopamine averaged over MD snapshots recorded in the time interval 50–100 ns.
-
†
RMSDs (root-mean-square-deviations) in VMAT2 Cα-atoms positions; all averages and standard deviations refer to the portion 50–100 ns of MD simulations.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Human vesicular monoamine transporter 2 | Clone ID 40025175 | NCBI Reference Sequence: BC108928 | Horizon Discovery |
| Gene (Lama glama) | Anti-GFP nanobody | Plasmid #49172 | Addgene | |
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | Cat # ATCC CRL-3022 | Used for expression of VMAT2 (PMID:27929454) |
| Cell line (Spodoptera frugiperda) | Sf9 | ATCC | Cat # ATCC CRL-1711 | Used in production of baculovirus for transduction (PMID:27929454) |
| Transfected construct (human) | pEG BacMam | Gouaux lab | PMID:25299155 | |
| Affinity chromatography resin | Strep-Tactin Superflow high capacity resin | IBA Lifesciences | Cat # 2-1208-500 | Affinity purification resin |
| Chemical compound, drug | n-Dodecyl-β-D-maltoside | Anatrace | Cat # D310 | Detergent |
| Chemical compound, drug | Cholesteryl hemisuccinate | Anatrace | Cat # CH210 | Lipid |
| Chemical compound, drug | Reserpine | Sigma | Cat # 83580 | Inhibitor |
| Chemical compound, drug | Dihydrotetrabenazine | Cayman Chemicals | Cat # 27182 | Inhibitor |
| Chemical compound, drug | [3H]5-HT | PerkinElmer | Cat # NET1167250UC | Radiolabeled substrate |
| Chemical compound, drug | [3H]dihydrotetrabenazine | American Radiolabeled Chemicals | Cat # ART 2119 | Radiolabeled inhibitor |
| Chemical compound, drug | Tetrabenazine | Sigma | Cat # T2952 | Inhibitor |
| Chemical compound, detergent | Digitonin | Sigma | Cat # 300410 | Detergent |
| Software, algorithm | Phenix | PMID:22505256 | RRID:SCR_014224 | https://www.phenix-online.org/ |
| Software, algorithm | SerialEM | PMID:16182563 | RRID:SCR_017293 | http://bio3d.colorado.edu/SerialEM |
| Software, algorithm | CryoSPARC | PMID:28165473 | RRID:SCR_016501 | https://cryosparc.com/ |
| Software, algorithm | RELION | PMID:23000701 | RRID:SCR_016274 | http://www2.mrc-lmb.cam.ac.uk/relion |
| Software, algorithm | UCSF-Chimera | PMID:15264254 | RRID:SCR_004097 | https://www.cgl.ucsf.edu/chimera/ |
| Software, algorithm | Coot | PMID:15572765 | RRID:SCR_014222 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot |
| Software, algorithm | MolProbity | PMID:20057044 | RRID:SCR_014226 | http://molprobity.biochem.duke.edu/ |
| Other | R 2/1 200 mesh Au holey carbon grids | Electron Microscopy Sciences | Cat # Q210AR1 | Cryo-EM grids |
| Other | R 1.2/1.3 200 mesh Cu holey carbon grids | Electron Microscopy Sciences | Cat # Q2100CR1.3 | Cryo-EM grids |
| Other | Copper HIS-Tag YSI | PerkinElmer | Cat # RPNQ0096 | SPA beads |
