Drug inhibition and substrate transport mechanisms of human VMAT2

Wei, Feiwen; Liu, Huihui; Zhang, Wei; Wang, Jufang; Zhang, Yanqing

doi:10.1038/s41467-024-55361-0

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Published: 02 January 2025

Drug inhibition and substrate transport mechanisms of human VMAT2

Nature Communications volume 16, Article number: 323 (2025) Cite this article

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Abstract

Vesicular monoamine transporter 2 (VMAT2) is crucial for packaging monoamine neurotransmitters into synaptic vesicles, with their dysregulation linked to schizophrenia, mood disorders, and Parkinson’s disease. Tetrabenazine (TBZ) and valbenazine (VBZ), both FDA-approved VMAT2 inhibitors, are employed to treat chorea and tardive dyskinesia (TD). Our study presents the structures of VMAT2 bound to substrates serotonin (5-HT) and dopamine (DA), as well as the inhibitors TBZ and VBZ. Utilizing cryo-electron microscopy (cryo-EM), mutagenesis functional assays, and molecular dynamics (MD) simulations, we elucidate the mechanisms of substrate transport and drug inhibition. Our MD simulations indicate potential binding poses of substrate (5-HT) in both cytosol-facing and lumen-facing states, emphasizing the significance of protonation of key acidic residues for substrate release. We demonstrate that TBZ locks VMAT2 in a lumen-facing occluded state, while VBZ stabilizes it in a lumen-facing conformation. These insights enhance our understanding of VMAT2 function and provide valuable insights for the development of novel therapeutic strategies for psychiatric disorders.

Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines

Article Open access 02 January 2024

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2

Article 01 November 2023

Transport and inhibition mechanisms of human VMAT2

Article 11 December 2023

Introduction

Vesicular amine transporters, also known as solute carrier 18 (SLC18) family members, are responsible for the loading and packaging of amine neurotransmitters into synaptic vesicles¹. This family comprises vesicular monoamine transporters 1 and 2 (VMAT1/2, encoded by SLC18A1/2), vesicular acetylcholine transporter (VAChT, encoded by SLC18A3), and vesicular polyamine transporter (VPAT, encoded by SLC18B1) (Supplementary Fig. 1d). These transporters are pivotal for the precise regulation of neurotransmitters packaging, a process closely linked to their subsequent release into the synaptic cleft. VMAT1/2 specifically handles various monoamine neurotransmitters, including 5-HT, DA, norepinephrine (NE), epinephrine, and histamine, all of which play significant roles in physiological processes like emotion, cognition, motor control, and learning^2,3,4. VMAT1 is mainly found in the large dense-core vesicles of neuroendocrine cells, while VMAT2 is predominantly expressed in monoaminergic neurons throughout the central, peripheral, and enteric nervous systems^5,6. Dysfunction of VMAT2 has been implicated in a variety of neurological and neuropsychiatric disorders, such as schizophrenia, mood disorders, and Parkinson’s disease⁷.

In the central nervous system, VMAT2 resides on synaptic vesicles and acts as a secondary active transporter. It depends on a proton electrochemical gradient directed toward the cytoplasm, created by the vacuolar-type H⁺-ATPase (V-ATPase), which maintains a lumen pH of approximately 5.7⁸. The transport of each cytosolic monoamine neurotransmitter into the synaptic vesicle involves the exchange of two luminal protons.

Dopamine dysregulation is closely linked to various movement disorders. Excessive dopaminergic activity can lead to hyperkinetic disorders such as tremors, tics, myoclonus, dystonia, and chorea, while reduced activity is associated with hypokinetic conditions, including Parkinson’s disease^9,10,11. Given VMAT2’s critical role in dopamine regulation, inhibiting its activity represents a promising therapeutic strategy. TBZ, the first VMAT2 inhibitor approved by FDA, was sanctioned for the treatment of chorea associated with Huntington’s disease in 2008 and has also been used off-label to manage TD, a severe side effect of antipsychotic medications linked to dopamine receptor hypersensitivity¹². However, TBZ has a short serum half-life and is rapidly metabolized into various stereoisomers, some of which may cause off-target side effects like akathisia, somnolence, depression, and suicidality¹³. In 2017, two TBZ derivatives, deutetrabenazine and VBZ, received FDA approval for TD treatment, both exhibiting a reduced risk of adverse effects^14,15. VBZ undergoes unique metabolic conversion to (+)-α-dihydrotetrabenazine (HTBZ) after the cleavage of its valine moiety, circumventing troublesome enantiomers and enabling once-daily dosing due to its longer half-life¹⁶.

Despite their clinical relevance, the mechanisms governing VMAT2 transport and inhibition remain inadequately understood, primarily due to the absence of structural data detailing VMAT2 in complex with its substrates or inhibitors. In this study, we present cryo-EM structures of human VMAT2 bound to the substrates 5-HT and DA, alongside the inhibitors TBZ and VBZ. By integrating these structural insights with MD simulations and functional mutagenesis studies, we clarify the substrate release mechanism of VMAT2 and the action mechanisms of drugs targeting TD, contributing to the advancement of neurological therapies centered on VMAT2.

Results

Structure determination

Human VMAT2 is a compact membrane protein that lacks extramembrane features, complicating structure determination via cryo-EM techniques. To address this challenge, we employed a methodology described in the literature¹⁷. We fused a BRIL tag to the N-terminus of VMAT2_12-514 (designated VMAT2_EM) and incubated the resulting protein with anti-BRIL Fab and anti-Fab nanobody to enhance particle size (Supplementary Fig. 1a, b). To assess the functionality of this fusion protein, we measured its transport activity for the fluorescent substrate FFN206 in HEK293T cells¹⁸. Transfected HEK293T cells effectively allow FFN206 to accumulate in acidic compartments via VMAT2, where V-ATPase provides the necessary proton gradient for transport. The results indicated that the transport activity of VMAT2_EM is approximately 60% of that observed with wild-type VMAT2 (VMAT2_WT). Additionally, VMAT2_EM demonstrated sensitivity to the inhibitors TBZ and VBZ, with FFN206 transport completely inhibited by both compounds, similar to VMAT2_WT (Fig. 1a). We also evaluated concentration-dependent inhibition of FFN206 uptake by TBZ in both VMAT2_WT and VMAT2_EM in HEK293T cells. The IC₅₀ values were comparable: VMAT2_WT showed 20.4 ± 4.1 nM, while VMAT2_EM had a value of 22.5 ± 6.3 nM (Fig. 1b), aligning well with previous studies¹⁹. Therefore, VMAT2_EM effectively captures the transport and drug inhibition profiles seen in VMAT2_WT.

We successfully solved the structures of VMAT2_EM bound to TBZ, VBZ, 5-HT, and DA at 3.28, 3.38, 2.98, and 3.00 Å, respectively (Fig. 1c–e, Supplementary Figs. 2–5 and Supplementary Table 1). Although there were no clear densities for the BRIL, Fab, or nanobody, the cryo-EM densities associated with the compounds were well-defined, allowing for reliable ligand assignments supported by ligand-free docking and MD simulations (Figs. 2a, d, 3a, d, Supplementary Fig. 6 and Supplementary Table 2).

**Fig. 2: Inhibition mechanisms of VMAT2 by TBZ and VBZ.**

**Fig. 3: Substrate binding mechanisms of 5-HT and DA.**

Structural analyses reveal that VMAT2 adopts a compact major facilitator superfamily (MFS) fold²⁰, characterized by 12 transmembrane helices (TMs) arranged into two pseudo-symmetrical domains: the N-terminal domain (NTD, TMs 1–6) and the C-terminal domain (CTD, TMs 7–12), both of which are oriented toward the cytoplasm. The luminal loop between TM1 and TM2 (LL1-2) exhibited flexibility and remained unresolved (Supplementary Fig. 1c). Notably, VBZ, 5-HT, and DA bind to VMAT2 in a lumen-facing conformation, whereas TBZ appears to trap VMAT2 in a lumen-facing occluded state (Fig. 1d, e). In these structures, the cytoplasmic gate is effectively sealed by two layers of residues: the inner layer consists of M204, M221, M403, and Y422, while the outer layer includes two salt bridges, D214-R357 and R217-D411 (Supplementary Fig. 7d, f). A previous study showed that mutating R218 in rat VMAT2 (corresponding to R217 in humans) enabled reserpine (RES) binding in the absence of a proton gradient, indicating that disruption of this salt bridge results in a conformation facing the cytosol²¹.

Locking VMAT2 in different conformations by TBZ and VBZ

To elucidate the inhibition mechanisms of TBZ and VBZ on VMAT2, particularly in the context of treatment of TD, we determined the cryo-EM structures VMAT2-TBZ and VMAT2-VBZ complexes. The structures reveal that TBZ traps VMAT2 in a lumen-facing occluded state, rendering it inaccessible from either side of the membrane (Fig. 2b and Supplementary Fig. 7a, c, d). On the luminal side, W318 from the luminal loop connecting TM7 and TM8 (LL7-8), along with a series of hydrophobic residues—V41, P45, F135, and F334—acts as a plug, effectively blocking the luminal gate (Fig. 2g and Supplementary Fig. 7c). TBZ occupies the central cavity formed by TMs 1, 4, 7, and 10. The binding site for TBZ is predominantly composed of non-polar residues from both the NTD and CTD, including L37, I44, L228, V232, and I308, which align with the hydrophobic architecture of TBZ. F135 contributes to π-π stacking with the benzene ring of TBZ. Specifically, the highly conserved residue E312 forms a salt bridge with the positively charged tertiary amine of TBZ, positioned roughly in the center of the molecule, aligning with previous findings that the equivalent residue E313 in rat VMAT2 is critical for both inhibitor recognition and substrate uptake²². R189 forms a polar interaction with one of TBZ’s methoxy groups, further stabilizing its binding (Fig. 2c). Notably, TBZ selectively inhibits VMAT2 rather than VMAT1²³, likely due to differences in the residues at the binding sites, such as L37, V232, I308, and Y433 in VMAT2 (Supplementary Fig. 1d). The structure indicates that TBZ’s inhibition mechanism may involve inducing an intermediate occluded conformation of VMAT2, disrupting the transport cycle.

As a derivative of TBZ, VBZ is the valine ester of (+)-α-HTBZ, the most active metabolite of TBZ (Fig. 1c)²⁴. Unlike TBZ, VBZ captures VMAT2 in a lumen-facing state (Fig. 2e). The cytoplasmic gate aligns similarly to that of VMAT2-TBZ, while the luminal gate remains open (Fig. 2g and Supplementary Fig. 7a). VBZ binds to the central cavity similarly to TBZ, involving numerous overlapping non-polar residues that contribute to hydrophobic interactions. E312 also forms a salt bridge with VBZ’s positively charged amine. The density reveals that R189 retains weak interaction with VBZ, with additional hydrogen bond interactions between the valine moiety and Y341 and Y433 (Fig. 2f). However, structural superposition indicates that VBZ shifts approximately 1.2 Å towards the lumen compared to TBZ, likely due to the steric effects of its additional valine moiety (Fig. 2g). These differences in binding sites between VBZ and TBZ may explain the distinct conformations observed in VMAT2.

Upon superposing the structures of VMAT2-TBZ and VMAT2-VBZ, we observed a contraction in TMs 2, 8, and 10 in VMAT2-TBZ. Notably, the C-terminus of TM7 transitions from a helical structure to a loop, causing W318 to reposition and block the luminal opening. This transition coincides with a dilation of TM11 and the emergence of luminal loop 1–2, which inserts between LL7-8 and TM11 (Fig. 2g). The distinct conformations induced by VBZ and TBZ suggest different inhibition mechanisms: VBZ stabilizes VMAT2 in a lumen-facing state, while TBZ induces changes leading to a lumen-facing occluded state, obstructing the transport cycle at various stages. Notably, VBZ is metabolized into (+)-α-HTBZ following the cleavage of its valine moiety after oral administration²⁵. This metabolite may further sustain an inhibitory role by promoting a lumen-facing occluded state, in line with VBZ’s longer half-life and the suitability for once-daily dosing.

Substrate binding sites of 5-HT and DA

To investigate the transport mechanism of monoamine neurotransmitters by VMAT2, we analyzed cryo-EM structures of VMAT2 bound to 5-HT and DA. 5-HT was observed in a lumen-facing conformation (Fig. 3b and Supplementary Fig. 7e, f). The cryo-EM density suggests that the amine group of 5-HT interacts with D399 (Pose_N-D399) (Fig. 1f), while ligand-free docking indicates coexistence of an alternate pose with the amine group directed at E312 (Pose_N-E312). MD simulations confirmed that 5-HT in Pose_N-D399 was stable, while Pose_N-E312 rapidly converted to Pose_N-D399 (Supplementary Fig. 6a, b). Thus, we confidently modeled 5-HT in the lumen-facing state as Pose_N-D399, where the positively charged amine forms a salt bridge with D399 and interacts with N305, Y341, and Y433. The hydroxyl group also engages in hydrogen bond with E312, while V232, I308, and F334 contribute to hydrophobic interactions with the indole group of 5-HT (Fig. 3c).

DA similarly binds to VMAT2 in a lumen-facing state (Fig. 3e, f and Supplementary Fig. 7b). The cryo-EM density for DA is well-defined and corroborated by ligand-free docking and MD simulations. Like 5-HT, MD simulations revealed that DA in Pose_N-D399 was stable, while Pose_N-E312 quickly transitioned to Pose_N-D399 (Supplementary Fig. 6c, d). Consequently, we modeled DA in Pose_N-D399. Its binding sites closely resemble those of 5-HT, with two hydroxyl groups interacting with E312 and S338, respectively (Fig. 3f).

Cell-based fluorescent uptake assays using FFN206 in HEK293T cells showed that mutations at N305, E312, Y341, and D399 significantly decreased transport activity, underscoring their critical roles in substrate transport (Fig. 3h and Supplementary Figs. 8, 9). These observations align well with previous studies^21,22. Despite forming a hydrogen bond with the substrate’s amine, the Y433A mutation exhibited a moderate decrease in transport activity compared to Y341A, likely due to the loss of a cation-π interaction. Given the shared characteristics of substrates for vesicular monoamine transporters, the highly conserved residues E312 and D399 likely play pivotal roles in the transport of monoamine neurotransmitters (Supplementary Fig. 1).

The binding sites of TBZ and VBZ overlap significantly with those of 5-HT and DA. Structural superposition indicates that the isobutyl group of TBZ or VBZ occupies a region similar to the indole group of 5-HT and the benzene ring of DA (Fig. 3g), revealing a steric overlap characteristic of these two inhibitors.

MD simulations for potential protonation sites

The proton gradient toward the cytosol facilitates the transition of VMAT2 from the lumen-facing state to the cytosol-facing state^26,27, suggesting potential protonation of acidic residues in the translocation pathway. We calculated the pKa values of acidic amino acids along the substrate pathway in lumen-facing VMAT2, identifying D399, D426, and E312 as possible protonation sites (Fig. 4a).

**Fig. 4: Molecular dynamics (MD) simulations of lumen-facing VMAT2-5-HT structure under different protonation conditions.**

To further explore the binding of 5-HT under these conditions, we conducted MD simulations for various protonation states: E312, D399, E312-D399, D399-D426, and E312-D399-D426. Our results revealed that protonation of D399 redirects the amine group of 5-HT toward E312, while simultaneous protonation of E312, D399, and D426 facilitates rapid substrate release (Fig. 4b–g). To validate these findings, we performed transport uptake assays with mutagenesis, substituting each amino acid with arginine, alanine, or asparagine/glutamine. All mutants exhibited significantly reduced transport activity, highlighting the critical roles of these residues in substrate transport, consistent with prior studies (Fig. 4h and Supplementary Figs. 8, 9)²².

Based on the established mechanism where one cytosolic transmitter is exchanged for two luminal protons²⁷, previous research indicated that one proton aids in substrate translocation while the other facilitates conformational transition^26,27,28. Our results suggest that the protonation of D399, E312, and D426 are linked to rapid substrate release. Given the higher pKa values of D399 and D426 compared to E312, we speculate that the proton from E312 dissociates into the lumen, while those from D399 and D426 are released into the cytosol (Fig. 4i). Although D426 does not directly bind substrates, it participates in a hydrogen bond network that is stronger in the lumen-facing state and weaker in the cytosol-facing state (Supplementary Fig. 10). This suggests that, in addition to its role in substrate release observed in our MD simulations, D426 may also play a crucial role in conformational transitions, aligning with previous studies that proposed a hinge mechanism involving the corresponding residue D427 in rat VMAT2^22,29.

Discussion

Our study elucidates the cryo-EM structures of human VMAT2 bound to the neurotransmitters 5-HT and DA in a lumen-facing state, as well as the clinical drugs TBZ and VBZ in occluded and lumen-facing states, respectively. While preparing this manuscript, we noted significant advancements in VMAT2 structural studies. Coleman et al. reported³⁰ the structure of VMAT2 bound to TBZ, while Lee et al. resolved³¹ various states of VMAT2 with ligands such as TBZ, RES, and 5-HT. Jiang et al. published^32,33 structures of VMAT2 with multiple ligands, including 5-HT, RES, TBZ, and others. Qu et al. and Iwata et al. detailed^34,35 VMAT2 structures with various ligands, and Zhou et al. presented³⁶ structures of frog VMAT2 and engineered sheep VMAT2 adopting a non-canonical fold.

Our structures of 5-HT- and DA-bound VMAT2, along with the TBZ-bound occluded state, align with recent findings, showing similar ligand poses. Notably, we provide the structure of VMAT2 bound to VBZ. Collectively, our findings and existing studies clarify substrate binding and transport mechanisms, highlighting diverse inhibition mechanisms. For instance, RES locks VMAT2 in a cytosol-facing state^31,32,34, TBZ traps it in an occluded state^{30,31,32,34,35}, and ketanserin stabilizes VMAT2 in a partially lumen-facing state³². Our study establishes a lumen-facing VBZ-bound state, revealing various inhibition mechanisms that enhance our understanding of drug actions.

Among the recently reported VMAT2 structures, only two cytosol-facing structures bound to substrates—5-HT and NE—have been resolved through site mutations, with their binding poses completely flipped: 5-HT in Pose_N-E312³¹ and NE in Pose_N-D399³³. To clarify the binding pose of 5-HT in the cytosol-facing state, we conducted MD simulations, indicating that 5-HT flips between poses, suggesting both orientations may coexist (Supplementary Fig. 11). These reported structures, along with our MD results, imply that substrate loading onto VMAT2 from the cytosol may occur in different orientations influenced by substrate type.

Some recent studies on VMAT2 have incorporated MD simulations to explore the protonation impact on transport. Coleman et al. found³⁰ that in the occluded state of DA-bound VMAT2, protonation of D399 directs the nitrogen of DA toward E312, while complete substrate release requires protonation of D33, E312, D399, and D426. Qu et al. conducted³⁴ MD simulations in a lumen-facing state with 5-HT, showing that protonation of D399 causes the nitrogen of 5-HT to orient toward E312, and that protonation of both D399 and E312 facilitates rapid substrate release, differing from our results. Jiang et al. also performed³² MD simulations for VMAT2-5-HT in the lumen-facing state, focusing primarily on the protein backbone. Variations in MD outcomes may arise from different methodologies or subtle conformational changes due to protein modifications. Nonetheless, a common observation is that protonation of D399, which directly interacts with the substrate’s amine group, is likely critical for substrate release, indicating a coupling mechanism between protonation and substrate release. Although the MD simulations in Coleman’s study³⁰ involved a different VMAT2 state, their findings largely align with ours, emphasizing D426 as another key protonation site. Additionally, as noted in Jiang’s work^32,33, D33, like D426, is another critical residue that does not participate in substrate binding but is part of the same broad hydrogen bond network, which may be involved in protonation (Supplementary Fig. 10). Further investigations are needed to deepen our understanding of the protonation mechanism of VMAT2.

In addition to our structural analyses of VMAT2 in complex with 5-HT and DA, we present the structure of VMAT2 bound to VBZ and highlight the distinct inhibition mechanisms of the FDA-approved drugs TBZ and VBZ on VMAT2, which have important implications for treating TD. While TBZ primarily acts as a reuptake inhibitor, VBZ’s unique mechanism may offer alternative therapeutic benefits by stabilizing different conformational states of VMAT2. We also investigated the binding poses of 5-HT in both the cytosol-facing and lumen-facing states through comprehensive MD simulations, underscoring the role of protonation on key acidic residues. These findings significantly enhance our understanding of VMAT2 function and offer valuable insights for drug development targeting psychiatric disorders.

Methods

Expression and purification of BRIL-VMAT2

cDNA of human VMAT2 (UniProt ID: Q05940) gene was synthesized and cloned with N-terminal BRIL sequence into a pCAG vector containing C-terminal tandem His and Flag affinity tags (BRIL-VMAT2_12-514-His-Flag). The plasmid containing BRIL sequence was donated by Z. Zhang group, as described in a previous study³⁷. The detailed protein sequence is provided in the Source Data in this paper. For the expression of VMAT2 protein, Expi293F cells were used and cultured in SMM 293T-II medium at 37 °C under 5% CO₂ at 130 rpm in a Multitron-Pro shaker. When the cell density reached approximately 2.0 × 10⁶ cells/mL, Expi293F cells were transiently transfected with the plasmid and polyethylenimines (PEIs) (Shanghai Maokang Biotechnology Co., Ltd.). The total amount of plasmids for transfection of one liter of cell culture is 2 mg and plasmids were pre-mixed with PEIs in 50 mL fresh medium in a 1:2 (w/w) ratio for 20 min before adding the mixture into cell culture.

After transfection for 48 h, cells were harvested by centrifugation at 1200 × g and resuspended in the buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 1.3 μg/mL aprotinin (Amresco), 0.7 μg/mL pepstatin (Amresco), and 5 μg/mL leupeptin (Amresco). The membrane fraction was extracted with 1% (w/v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) at 4 °C for 2 h. After extraction, the cell lysate underwent centrifugation at 12,000 × g for 1 h (JA 25.50, Beckman Coulter) and supernatant was loaded to anti-Flag M2 affinity resin (GenScript), washed in buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.01% LMNG and 0.001% CHS (w/v), and eluted with buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.01% LMNG and 0.001% CHS (w/v) and 0.4 mg/mL Flag peptide. The elution was then loaded to nickel affinity resin (Ni-NTA, Cytiva), washed with the wash buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.01% LMNG and 0.001% CHS (w/v), 20 mM imidazole. The protein was eluted with the wash buffer plus 300 mM imidazole. The protein sample after elution was collected and concentrated to 5 mg/mL using a 50 kDa molecular weight cutoff concentrator (Millipore).

Expression and purification of anti-BRIL Fab

The plasmid containing anti-BRIL Fab sequence, including heavy chain and light chain, was donated by Z. Zhang group, as described in a previous study³⁷. The detailed protein sequence is provided in the Source Data in this paper. DNA sequences of the heavy and light chains of anti-BRIL Fab were cloned into pCAG expression vectors separately. A His-tag was attached to the C-terminal of the heavy chain. For the expression of Fab, Expi293F cells were cultured and co-transfected by plasmids of heavy chain and light chain under the same conditions as VMAT2. The total amount of heavy chain and light chain plasmids for transfection of one liter of cell culture is 2 mg, 1 mg of each.

Purification of anti-BRIL Fab was conducted as described previously with some modifications³⁷. Briefly, after transfection for 48 h, the cell culture media was centrifuged at 1200 × g for 10 min, and then the supernatant was loaded to nickel affinity resin, washed with the wash buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 50 mM imidazole. The protein was eluted with the wash buffer plus 300 mM imidazole. The protein sample after elution was collected and concentrated to 2 mg/mL using a 50 kDa molecular weight cutoff concentrator (Millipore).

Expression and purification of anti-Fab nanobody

The plasmid containing anti-Fab nanobody sequence was donated by Z. Zhang group, as described in a previous study³⁷. The detailed protein sequence is provided in the Source Data in this paper. The DNA sequence corresponding to the anti-Fab nanobody (Nb) was inserted into the pET24(+) vector, which features an N-terminal PelB signal sequence and a C-terminal 6 × His-tag. This construct allowed for the expression and translocation of Nb to the periplasmic space of Escherichia coli strain BL21. The bacteria were cultured in LB medium at 37 °C until the optical density at 600 nm (OD₆₀₀) reached approximately 0.8. Subsequently, protein expression was induced by the addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and continued for 16 h at 16 °C.

For Nb purification, the bacterial cell pellets were lysed by sonication in lysis buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM PMSF). Cell debris was removed by centrifugation at 33,000 × g for 1 h. Then the supernatant was applied to Ni-NTA, washed with 25 mM HEPES pH 7.4, 150 mM NaCl, 25 mM imidazole and 50 mM imidazole separately. Elution of the protein was performed using an elution buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl and 500 mM imidazole. The protein sample after elution was collected and concentrated to 2 mg/mL using a concentrator with a molecular weight cutoff of 10 kDa (Millipore).

The BRIL-VMAT2/Fab/Nb complex assembly

For complex assembly, the purified BRIL-VMAT2, anti-BRIL Fab, and anti-Fab Nb were incubated on ice for 1 h at the ratio of 1:1.4:2. Then the samples were applied to a Superose 6 Increase 10/300 GL column (Cytiva) equilibrated with buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.01% LMNG and 0.001% CHS (w/v) to remove the excess Fab and Nb components. The peak fractions corresponding to the ternary complex were collected and concentrated to 10 mg/mL.

Cryo-EM sample preparation and data acquisition

The final concentration of protein samples for cryo-EM sample preparation is ∼10 mg/mL. For VMAT2-5-HT/TBZ/VBZ/DA, compounds of serotonin (5-HT, Target-Mol), tetrabenazine (TBZ, Target-Mol), valbenazine (VBZ, MedChemExpress), and dopamine (DA, MedChemExpress) were added to the protein solution at the final concentration of 2.5 mM, 100 μM, 15 μM, and 6.6 mM, individually. 3.5 μL of protein sample with compounds was applied to a glow-discharged Quantifoil R1.2/1.3 400-mesh gold holey carbon grid, blotted for 4.5 s and flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot (Mark IV, Thermo Fisher Scientific).

For data acquisition of VMAT2-5-HT/TBZ/VBZ/DA, micrographs were acquired on a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV with a Falcon 4i Direct Electron Detector (Thermo Fisher Scientific) and a Selectris X energy filter. EPU was used for automated data collection following standard procedures. A nominal magnification of 130,000 × was used for imaging, yielding a pixel size of 0.932 Å on images. The defocus range was set from −0.9 μm to −1.6 μm. Each micrograph was recorded in a total dose of ∼50 e⁻/Å², with total collection of 13,925, 23,115, 13,947, and 19,844 movie stacks, respectively.

Cryo-EM data processing

The data processing pipelines are shown in Supplementary Figs. 2–6. Motion correction was performed using Relion v3.1³⁸ and CTF estimations were performed using cryoSPARC v3.3.2³⁹. For the data processing of VMAT2-5-HT, after several rounds of 2D classification, an initial model with clear TMs was obtained using cryoSPARC. Heterogeneous Refinement, Local refinement and Local CTF refinement yielded a lumen-facing VMAT2-5-HT at the resolution of 2.98 Å. Similarly, we got an occluded VMAT2-TBZ at the resolution of 3.28 Å, a lumen-facing VMAT2-VBZ at the resolution of 3.38 Å, and a lumen-facing VMAT2-DA at the resolution of 3.00 Å.

Model building

We first built the protein atomic model of VMAT2-5-HT based on the AlphaFold⁴⁰ predicted model as a starting template. Using UCSF Chimera⁴¹ to dock this model into the density maps. Atomic model building based on the density map of VMAT2-5-HT was performed in Coot⁴², and refined in real space using Phenix.real_space_refine⁴³. Validation tools in Phenix⁴³ and Molprobity⁴⁴ were used to guide iterative rounds of model adjustment in Coot and refinement in Phenix. For model building of VMAT2-DA/VBZ, atomic model of VMAT2-5-HT was used as initial model, because of the similar conformations. As for VMAT2-TBZ, the AlphaFold predicted model was used as the starting template, and modified subsequently according to the density map.

As regards the model building of the ligands, we conducted ligand-free docking and calculated the binding free energies through the molecular mechanics-generalized born surface area (MM/GBSA) approach by employing the Schrödinger Maestro program (Maestro, Schrödinger, LLC, New York, NY, 2024) for ligand binding pose determination. Consequently, we constructed the ligand models by referencing to the EM density map and the adjacent environment with Coot. These ligand models underwent further refinement with Phenix.real_space_refine and validated by MD simulations. Visualization of all figures was accomplished using PyMOL (www.pymol.org) or UCSF ChimeraX⁴⁵.

FFN206 uptake assay using HEK293T cells

Uptake assays were performed using fluorescent substrate FFN206 in HEK293T cells as reported previously¹⁸, with some modifications. Briefly, HEK293T cells were cultured in 6-well plates until reaching approximately 50–60% confluence, and expression plasmids pCAG-WT or pCAG-mutants were transfected with PEIs following the manufacturer’s instructions. After transfection for 48 h, the medium was discarded, and cells were incubated with 2 mL assay buffer (125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM NaH₂PO₄, 1.2 mM MgSO₄, 1.3 mM CaCl₂, 5.6 mM glucose and 25 mM HEPES pH 7.4) at 37 °C for 15 min. Subsequently, the fluorescent indicator substrate FFN206 (Target-Mol) was added to the cells at a final concentration of 5 μM in assay buffer, and the cells were incubated at 37 °C for 1 h. Uptake was terminated by washing cells three times with 2 mL ice-cold phosphate-buffered saline (PBS). Cells were lysed with PBS containing 1% TritonX-100, and the fluorescence intensity of FFN206 was measured with excitation/emission wavelength at 360 nm/460 nm using a microplate reader (BioTek Synergy2 Microplate Reader). All statistical analyses were performed with one-way ANOVA followed by Dunnett’s multiple comparisons test by comparing WT with mutants using GraphPad Prism v9.3.1. Transport assays were biologically repeated at least three times. The average fluorescent signals of untransfected cells were treated as background correction and the average fluorescent signals of VMAT2_WT were defined as 100% uptake.

Inhibition of VMAT2-mediated uptake of FFN206 by compounds of TBZ/VBZ

For the concentration-dependent inhibition of FFN206 uptake assay, HEK293T cells were cultured in 12-well plates, until reaching approximately 50–60% confluence, and expression plasmids pCAG-VMAT2_WT or pCAG-VMAT2_EM were transfected with PEIs following the manufacturer’s instructions. After transfection for 48 h, the medium was discarded, and cells were treated with assay buffer containing the two compounds in six concentrations of 0.1–10,000 nM for TBZ or DMSO (as control), in the total volume of 1 mL, and incubated at 37 °C for 15 min. Next, cells were incubated for another 1 h in uptake buffer (assay buffer containing FFN206, final concentration 5 μM) at 37 °C and washed three times with 2 mL ice-cold PBS. Cells were lysed using PBS containing 1% TritonX-100 and transformed to a 96-well plate, the fluorescence intensity of FFN206 was measured with excitation/emission wavelength at 360 nm/460 nm using a microplate reader. The concentration-dependent inhibition of FFN206 uptake assays were biologically repeated at least three times. The average fluorescent signals of untransfected cells were treated as background correction and the average fluorescent signals of VMAT2_WT or VMAT2_EM were defined as 100% uptake separately. Curves were fitted using nonlinear regression with GraphPad Prism v9.3.1.

For the transport inhibition assay, HEK293T cells were cultured in 6-well plates, until reaching approximately 50–60% confluence, and expression plasmids pCAG-VMAT2_WT or pCAG-VMAT2_EM were transfected with Lipofectamine^TM 2000 according to the manufacturer’s instructions. After transfection for 24 h, the medium was discarded and cells were divided into 24-well plates. 10 mM sodium butyrate was added, and cells were cultivated at 30 °C for 24 h. Afterward, cells were treated with assay buffer containing 10 mM Glyco-diosgenin (GDN) with inhibitors (TBZ or VBZ) or DMSO (as control), and incubated at 37 °C for 15 min. Next, cells were incubated for another 1 h in uptake buffer (assay buffer containing FFN206, final concentration 5 μM) at 37 °C and washed three times with 2 mL ice-cold PBS. Cells were lysed using PBS containing 1% TritonX-100 and transformed to a 96-well plate, the fluorescence intensity of FFN206 was measured with excitation/emission wavelength at 360 nm/460 nm using a microplate reader (BioTek Synergy2 Microplate Reader). The inhibition assays were biologically repeated at least three times. In this assay, 5 μM TBZ and 5 μM VBZ were applied as inhibitors. The average fluorescent signals of untransfected cells were treated as background correction and the average fluorescent signals of VMAT2_WT were defined as 100% uptake.

Cell surface biotinylation and immunoblot analysis

Cell surface biotinylation and immunoblot analysis were performed as reported previously with some modifications⁴⁶. Briefly, HEK293T cells were cultured in 6-well plates until reaching approximately 70% confluence, and expression plasmids pCAG-WT or pCAG-mutants were transfected with PEIs following the manufacturer’s instructions. After 48 h, the cell culture medium was removed and cells were suspended with 1 mL ice-cold PBS and then treated with 1 mL sulfo-N-hydroxysuccinimide-SS-biotin (1 mg/mL in PBS, Thermo Scientific) at room temperature for 1 h. Afterward, cells were washed three times with 1 mL ice-cold PBS containing 100 mM glycine and incubated at 4 °C for 10 min in the same buffer. Then, cells were lysed with 500 μL lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, and 1% Triton X-100, containing protease inhibitors) at 4 °C for 2 h with shaking. After centrifugation at 13,000 × g for 10 min, the supernatants were incubated with 50 μL streptavidin-agarose beads (Thermo Scientific) at room temperature for 1 h. The beads were then centrifuged at 500 g for 1 min, washed three times with ice-cold lysis buffer, and incubated with 100 μL 2 × Laemmli buffer containing 100 mM dithiothreitol at room temperature for 1 h to recover the cell surface proteins. Samples were separated using SDS-polyacrylamide gel electrophoresis followed by western-blot analysis. All proteins were detected using HRP-conjugated DYKDDDDK tag monoclonal antibody (1:5000 dilution; Proteintech; HRP-66008). Then the same membrane was stripped at room temperature for 1 h. Na⁺/K⁺-ATPase was used as loading control for normalization and was detected with rabbit anti-Na⁺/K⁺-ATPase monoclonal antibody (1:10,000 dilution, ABclonal; A11683) combined with secondary HRP goat anti-rabbit monoclonal antibody (1: 10,000 dilution; ABclonal; AS014).

Total expression levels detected by Western blot

Total expression levels of WT and mutants of VMAT2 were performed as previously described⁴⁷ with modifications. HEK293T cells were cultured in 6-well plates until reaching approximately 90% confluence, and expression plasmids pCAG-WT or pCAG-mutants were transfected with PEIs following the manufacturer’s instructions. After 48 h, The cell culture medium was removed and cells were suspended with 1 mL ice-cold PBS. Then, after centrifugation and discarding the supernatant, cells were lysed with 150 μL lysis buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1% DDM, 1.3 μg/mL aprotinin, 0.7 μg/mL pepstatin, and 5 μg/mL leupeptin) at 4 °C for 2 h with shaking. The cell extracts were centrifuged at 16,000 × g for 1 h before the supernatant was taken and mixed with SDS loading buffer. Then the mixtures were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose filter membrane and probed with HRP-conjugated DYKDDDDK tag monoclonal antibody (1:5000 dilution; Proteintech; HRP-66008) or β-Actin rabbit mAb (1:5000 dilution; ABclonal; AC038). The secondary antibodies was used HRP-conjugated goat anti-rabbit IgG (H + L) (1:5000 dilution; ABclonal; AS014). The membrane was visualized with the imaging system (ChemiDoc Touch; Bio-Rad, Hercules, CA, USA) using enhanced chemiluminescence (ECL) (Smart-Lifesciences).

Confocal imaging analysis

HEK293T cells were cultured in 20 mm glass bottom cell culture dishes (NEST, 801001) with 1.0 × 10⁶ cells/mL density overnight, and pCAG-GFP-VMAT2-His-Flag constructs were transfected with PEIs following the manufacturer’s instructions. 24 h post transfection, cells were observed under confocal microscope (Leica SP8).

Molecular dynamics simulations

MD simulations were conducted for VMAT2-5-HT/DA/TBZ/VBZ. The initial proteins include residues 18 to 46 and 117 to 475 for VMAT2-5-HT/VBZ, residues 18 to 46 and 132 to 475 for VMAT2-DA and residues 16 to 50, 117 to 318 and 324 to 476 for VMAT2-TBZ. The pKa values of protein residues were estimated using PROPKA 3.4.0⁴⁸. Charged residues were protonated assuming pH 7.4 unless otherwise specified, and all the N- and C-termini of proteins were capped with acetyl and N-methyl groups, respectively. To build the system, protein-ligand complex was firstly embedded in a POPC bilayer (~302 molecules) and then solvated in a water box (117 × 117 × 90 Å³). Finally, 150 mM NaCl was added to neutralize the system. Details of system composition are listed in Supplementary Table 2.

MD simulations were performed with GROMACS 2021.4⁴⁹. CHARMM36 force field was used for protein, POPC lipid and ions⁵⁰, and TIP3P model⁵¹ was used for water. The force fields of four ligands (5-HT/DA/TBZ/VBZ) were generated by CGenFF program⁵². After energy minimization by steepest descent algorithm, the systems were simulated in NVT ensemble to relax lipid tail, lipid head and solvent (water and ions) in steps for 1.5 ns. Then, the systems were further relaxed in NPT ensemble. Constraints on (1) lipids and solvent, (2) protein sidechain, (3) ligand, and (4) protein Cα atoms were gradually released each for 2 ns. After pre-equilibrations, 1 μs production simulations were run without any constraint. Periodic boundary condition was applied, and the temperature and pressure were kept at 310 K and 1 atm by Nose-Hoover thermostat⁵³ and Parrinello-Rahman barostat⁵⁴, respectively. Long range electrostatic interactions were described using the particle mesh Ewald (PME) method⁵⁵ with a cutoff of 12 Å. Van der Waals interactions were smoothly switched to zero from 10 to 12 Å. All bonds involving hydrogen atoms were constrained by the LINCS algorithm⁵⁶ to allow a time step of 2 fs. Each system was run for three replicas with different initial velocities and the total simulation time was 39 µs.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Information. Atomic coordinates of VMAT2-5-HT/DA/TBZ/VBZ have been deposited in the Protein Data Bank (http://www.rcsb.org) under the accession codes 8X3K, 9KQE, 9KQA, and 9KQ8. The corresponding electron microscopy maps have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/), under the accession codes EMD-38038, EMD-62501, EMD-62498, and EMD-62497. Source data are provided with this paper.

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Acknowledgements

We would like to thank the center of Cryo-Electron Microscopy, Core Facility of Shanghai Medical College, Fudan University. We thank the great support from Prof. Yanhui Xu in Fudan University. We thank Prof. Zhenguo Chen, Hui Zhao, Anqi Dong, Yulei Ren for cryo-EM facility support. We are grateful to Prof. Zhe Zhang from Peking University for kindly providing the plasmids of BRIL, Fab, and nanobody. This work was funded by the National Natural Science Foundation of China (32171196 to Y.Z.), National Key R&D Program of China (2021YFA1300101 to Y.Z.), Start-up funds from Fudan University and Shanghai Fifth People’s Hospital, and Shenzhen Natural Science Foundation Project (GXWD20201231105722002-20200831175432002 to H.L.).

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These authors contributed equally: Feiwen Wei, Huihui Liu, Wei Zhang.

Authors and Affiliations

Shanghai Fifth People’s Hospital, Fudan University, and Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Institutes of Biomedical Sciences, Fudan University, Shanghai, China
Feiwen Wei, Wei Zhang, Jufang Wang & Yanqing Zhang
Arieh Warshel Institute for Computational Biology, School of Medicine, The Chinese University of Hong Kong, Shenzhen, Guangdong, China
Huihui Liu

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Feiwen Wei
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Contributions

Y.Z. conceived the project. F.W. and J.W. prepared protein purification. W.Z. and F.W. performed functional transport assay. F.W. performed cryo-EM data acquisition. Y.Z. and F.W. contributed to the cryo-EM data processing and model building. H.L. performed MD simulations. Y.Z. wrote the manuscript. All authors analyzed the data and contributed to manuscript preparation.

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Correspondence to Yanqing Zhang.

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Wei, F., Liu, H., Zhang, W. et al. Drug inhibition and substrate transport mechanisms of human VMAT2. Nat Commun 16, 323 (2025). https://doi.org/10.1038/s41467-024-55361-0

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Received: 20 February 2024
Accepted: 10 December 2024
Published: 02 January 2025
DOI: https://doi.org/10.1038/s41467-024-55361-0