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VMAT2: a dynamic regulator of brain monoaminergic neuronal function interacting with drugs of abuse

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The authors report the cryo-EM structure of human vesicular monoamine transporter 2 VMAT2 bound to the noncompetitive inhibitor tetrabenazine in an occluded state. This important achievement captures the structure of an major facilitator superfamily MFS transporter that is critical for human neurotransmission. The evidence for the structure is solid , but there are several concerns regarding the mechanistic insights into the transport mechanism, which make the study more descriptive than explanatory. During the peer-review process the editor and reviewers write an eLife assessment that summarises the significance of the findings reported in the article on a scale ranging from landmark to useful and the strength of the evidence on a scale ranging from exceptional to inadequate. Learn more about eLife assessments. The vesicular monoamine transporter 2 VMAT2 is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Here we report a 3. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for intracellular and luminal gating, including a cluster of hydrophobic residues which are involved in a luminal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational change and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics. Neuronal signaling by monoaminergic neurotransmitters controls all aspects of human autonomic functions and behavior, and dysregulation of this leads to many neuropsychiatric diseases. Nearly 60 years ago 1 , 2 , secretory vesicles prepared from adrenal glands were shown to contain an activity that accumulated epinephrine, norepinephrine, and serotonin in an ATP-dependent manner 3. Extensive characterization by many different laboratories of synaptic vesicles SVs in neurons showed that monoamine transport activity was also dependent on the proton gradient generated by the V-ATPase, exchanging two protons for one cationic monoamine 4 — 8. Monoamine transport was shown to be inhibited by non-competitive inhibitors such as tetrabenazine TBZ 9 and competitive inhibitors like reserpine which has been used to treat hypertension 3. Amphetamines were shown to be monoamine transporter substrates, eventually leading to vesicle deacidification and dopamine release VMAT2 is expressed in all monoaminergic neurons in the brain, including those for serotonin, norepinephrine, dopamine, and histamine 3 , and is essential for loading these neurotransmitters into SVs. Sequence alignments also show that SLC18 transporters belong to the major facilitator superfamily MFS of membrane transport proteins which use an alternating access mechanism 27 , 28 to transport substrate across membranes. SLC18 members are predicted to be comprised of 12 transmembrane spanning helices TMHs , which are arranged in two pseudosymmetric halves each with 6 TMHs containing a primary binding site for neurotransmitters, polyamines, and inhibitors located approximately halfway across the membrane 29 Fig. Conformational changes driven by the proton electrochemical gradient are thought to expose the binding site to both sides of the membrane allowing for transport of neurotransmitter from the cytoplasm to the lumen of SVs 30 — The neurotransmitter substrate is bound at the central site yellow, triangle. The red and blue triangles depict the pseudo two-fold symmetric repeat comprised of TM and 7—12, respectively. Error bars show the s. Right panel, graphs of competition binding of 3 H-dihydrotetrabenazine with unlabeled reserpine, error bars show the s. The bars show the means and points show the value for each technical replicate. Small-molecule ligands such as TBZ and reserpine are high-affinity inhibitors of VMAT2, which prevent neurotransmitters from binding, arrest VMAT2 from cycling, and consequently reduce neuronal signaling. To overcome this, we incorporated mVenus and the anti-GFP nanobody into the N— and C-terminus respectively of human VMAT2 to provide mass and molecular features to facilitate the cryo-EM reconstruction, this created a hook-like fiducial feature by reconstituting the interaction of these proteins on the cytosolic side of VMAT2 Attachment of both proteins to the termini proved to be ineffective as the unstructured N— and C— terminus of VMAT2 are flexible; to combat this problem, we determined the minimal termini length that would reduce flexibility while maintaining VMAT2 folding. We investigated the consequences of modification of VMAT2 in order to ensure the chimera maintained functional activity. Next, we performed transport experiments using permeabilized cells, initial time course experiments using 3 H-labeled serotonin showed clear accumulation ED Fig. Thus, the functional properties of the chimera are similar to the wild-type VMAT2. The resulting cryo-EM map was determined to a resolution of 3. This demonstrates the feasibility of our approach and enabled us to build a model of VMAT2. TBZ is shown in light green sticks. Blue and red sticks denote residues in the N— and C-terminal half respectively. The blue dotted circle indicates the position of the hydroxyl group in dihydrotetrabenazine. TBZ is shown in light green sticks and the associated density in dark grey mesh. Green, red, and blue indicate hydrophobic, negative, or positively charged properties of the side chains. The cytosolic facing side of VMAT2 is characterized primarily by the unstructured N— and C-termini along with a residue loop that connects the two halves, extending from TM6 to TM7 before terminating in a short alpha helix that runs parallel to the bilayer and connects to TM7 with a short linker. TM5 and 11 both contain proline residues near the luminal face, which break the helical structure and facilitate connections to TM6 and 12 respectively. TM9 and 12 exhibit significant heterogeneity in our cryo-EM reconstructions, we speculate that this is likely due to a dynamic nature intrinsic to the TMs, an aspect that may offer a glimpse into VMAT2 dynamics. EL1 and 4 also contain intriguing elements of structure, EL1 extends into the luminal space in an unstructured loop which is mostly not resolved in our structure, before terminating in short helix which interacts with the luminal face of the transporter near TM7, 11 and A striking feature of EL4 is the location of W which positions its indole side chain directly into a luminal cavity near the TBZ site, acting as a plug to completely occlude the luminal side of the transporter. Together, these loops cinch the luminal side of the transporter closed, locking VMAT2 in an occluded conformation and preventing ligand egress. The conserved nature of EL4 and W suggests this motif is necessary for transport function and is a key player in the transporter mechanism ED Fig. The conformation of EL1 and 4 is likely also aided by a disulfide bond between cysteines and , which is known to be necessary for transporter function While our structure was not able to unequivocally place this bond due to the lack of density for residues of EL1, the disulfide likely restricts the movement of EL4 which orders this loop in a more rigid state. However, Alphafold lacks several key features such as in the position of the ELs and is unable to predict key details that are critical for ligand binding. Hence, computational docking could not capture a stable TBZ bound state using Alphafold, alluding to the critical importance of our experimental structure in understanding of VMAT2 molecular mechanisms. EL4 is involved in luminal gating and the PA variant would likely disrupt the conformation of the loop. In the case of proline , a histidine would result in not only an insertion of a positively charged residue into the luminal membrane interface but would also reduce the helical bend and distort the connection of TM5 with TM6. The PL variant would also disrupt helical connections and the overall architecture of the helix by insertion of a bulky residue into a small hydrophobic cavity. Therefore, these SNPs likely alter the ability of the transporter to sample multiple conformations by reducing transporter dynamics and also perturb VMAT2 folding. Recently, many additional disease variants have been discovered 47 many of which are also found in the luminal or cytoplasmic ends of TMHs, EL1, and the N— and C-termini. The structure of the VMAT2-TBZ complex reveals that both the cytosolic and luminal gates are closed which precludes solvent and ligand access from either the cytosolic or luminal compartments Fig. Previous studies have suggested residues R, M, Y and Y make up the cytosolic gate 32 , we find R and Y form the outer cytosolic gate with the guanidino group of R involved in a cation-pi interaction with the aromatic benzyl group of Y which seals off intracellular access to the binding site. It is likely that M and M also contribute to cytosolic gating in this region as their side chains also act to fill this space. On the luminal side, F, F and W, form the luminal gate where they interact with one another to block access to the binding site. E of EL1 may play a role in stabilizing the tryptophan in this conformation, with the carboxyl group of the side chain orienting itself near the indole nitrogen potentially forming a hydrogen bond pair. The large aromatic side chains of these residues may compartmentalize the transporter, ensuring directional transport of substrate. MD simulations revealed little variation in the pose of the aromatic gating residues comprising the inner gates Fig. However, the luminal gates showed more movement, likely owing to its residue composition and lack of strong interactions. Despite this observation, we would assert that the tight hydrophobic environment likely prevents exchange from the luminal space. These mechanisms of gating are atypical of MFS transporters which more commonly use salt bridges to gate access to the substrate binding site Upon careful inspection of the model, we were able to identify distinct polar networks that we believe may play a role in proton coordination and subsequent transporter conformational change. At the center of this network lies D33 32 , which makes critical contacts with the side chains of N34, K, S, and Q Together, the residues comprise a complex hydrogen bond network linking TM 1, 4 and D 49 lies further toward the cytosol with the side chain carboxyl group facing the bulk of the network, likely forming a hydrogen bond with the hydroxyl group of S In the other TMD half there are two distinct groups of interacting polar residues, which bridge between TM 7, 8 and 10 Fig 2e. The second group is a pair of residues found on the luminal side, between residues E and N with the amide group of the N side chain pointed towards the carboxyl group of E, which could act to stabilize TBZ in the binding site. The third group is located toward the cytosolic side and consists of N, Y, and D 49 , the latter two of which have previously been speculated to form a hydrogen bond pair The side chains of these residues are positioned toward one another, with the carboxyl group of D forming a hydrogen bond with N and likely Y Taken together, we believe these networks play a critical role in mediating conformation change in the transporter. We hypothesize that protonation of D33, E, and D would greatly perturb these interactions by breaking crucial hydrogen bond pairs, leading to opening of the cytosolic gate. The asymmetry between these two networks is also striking, with the first network consisting of TMs 1, 4 and 11 being substantially larger. This may allow for larger conformational changes on this side and an overall asymmetry in the cytosolic-open state of the transporter. To our knowledge this is an atypical feature in MFS proteins 48 and would represent an interesting adaptation upon the rocker-switch mechanism. TBZ Fig. The TBZ binding site exhibits an amphipathic environment, comprised of both polar and non-polar residues Fig. The tertiary amine of TBZ orients itself towards the negatively charged surface of the binding site near TMs 7 and 11, and toward E Fig. E plays an analogous role to the highly conserved aspartate residue present in neurotransmitter sodium symporters 50 which also is involved in directly binding to amine groups ED Fig. Because E was previously shown to be necessary for substrate transport and inhibitor binding, we first selected this residue for mutagenesis to probe its importance in TBZ binding 31 , Next, we observed that R orients the guanidino group towards the methoxy groups of TBZ likely forming hydrogen-bonding interactions and we found that replacement of R with an alanine nearly completely abolished DBTZ binding at all concentrations tested Fig. We speculate that R may also be involved substrate transport, by forming interactions with the hydroxyl groups of dopamine or serotonin ED Fig. Lysine , has been previously shown to play an important role in both TBZ binding and serotonin transport, and positions the primary amine side chain toward the methoxy groups of TBZ 49 Fig. K is positioned between two aspartate residues D and D33 and is part of a hydrogen bond network that has been previously hypothesized Previous experiments found that mutating K to alanine resulted in an approximate 4-fold reduction in TBZ binding affinity While significant, this did not abolish TBZ binding and K may play a more significant role in inducing conformational changes during proton transport rather than TBZ binding. Asparagine 34 is of particular interest since the amide group of the sidechain of N34 appears to form a hydrogen bond with the carbonyl oxygen of TBZ Fig 3b. Our structure suggests that the amide of N34 acts as a hydrogen bond donor for TBZ, and in the case of DTBZ the hydroxyl of the ligand may act as a hydrogen bond donor for the carbonyl oxygen of N We hypothesize that this interaction is more favorable for DTBZ, leading to a higher binding affinity. We hypothesize that N34 does not form a favorable hydrogen bond with the oxygen of valbenazine and that addition of this larger moiety causes steric clashes in the binding site. Large hydrophobic residues make a significant number of contacts with TBZ in the binding site and act as both space-filling residues and form critical aromatic interactions with the ligand. Extensive contacts of TBZ with F may function to keep the transporter closed on the luminal side, which would trap VMAT2 in the occluded conformation. Conversely, while W in EL4 also does not interact directly with TBZ, W is required for stabilizing the occluded conformation, and replacement with alanine prevents TBZ from being trapped inside the transporter by preventing closure of the luminal gate. Mutation of the disulfide bond between EL1 and 4 plays a critical role in transport 40 , and the disulfide may function to restrict the dynamics of this region to allow W to occlude the neurotransmitter binding site during transport. Previous studies have highlighted V 51 , which is a leucine in VMAT1, as being putatively involved in conferring differences in affinity, and our model shows that V is positioned closely to the isobutyl of TBZ which is wedged into a small hydrophobic pocket ED Fig. The addition of an extra carbon of the leucine sidechain would produce a steric clash and limit the ability of TBZ to bind Fig. The predominant pose in our simulations is identical to the pose resolved in our cryo-EM structure 0. We believe this pose provides insight into the mechanism by which TBZ enters and eventually positions itself into the pose resolved in our cryo-EM map. This result highlights the stepwise process inhibitors like TBZ undergo to bind their targets. Tetrabenazine green hexagon binds to the luminal-facing state and induces conformational change to a high-affinity occluded conformation which is the resolved cryo-EM structure reported in this work. VMAT2 functions by alternating access which involves exposure of the primary binding site to both sides of the membrane and isomerization between a cytosolic-open and luminal-open state in a mechanism known as the rocker-switch Fig 4a 3 , 29 , Since TBZ is a non-competitive inhibitor of neurotransmitter transporter, it enters VMAT2 from the luminal side, binding to a luminal-open conformation Fig 4a TBZ makes extensive contacts with residues in the primary site, likely in a lower affinity state as the transporter subsequently closes to form the high-affinity occluded state Fig. The luminal gates lock the transporter into an occluded state, preventing displacement by other ligands and producing a so-called dead-end complex 32 , 34 , 35 , Our structure also provides important clues for understanding the chemical specificity and selectivity of TBZ binding, suggesting that the enhanced affinity of DTBZ is due to preferential interaction with N We also highlight three key polar networks which may be involved in conformational changes induced by proton binding during the transport cycle and are likely also involved in mediating proton transduction. Thus, our work provides a framework for understanding the structural underpinnings of neurotransmitter transport and inhibition in VMAT2 and other related transport proteins. All authors contributed to editing and manuscript preparation. The data that support the findings of this study are available from the corresponding author upon request. The half-maps and masks used for refinement have also been deposited in the EMDB. The authors declare no competing interests. Correspondence and requests for materials should be addressed to coleman1 pitt. No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Enriched membranes were first isolated by sonication followed by an initial spin at 10,g followed by a ,g spin and subsequent homogenization. Membranes resuspended in 25 mM Tris pH 8. The VMAT2 chimera 1. Movies containing 40 frames were recorded on a FEI Titan Krios operating at kV equipped with a Gatan K3 direct electron detector and a Bioquantum energy filter set to a slit width of 20 eV. Images were collected in super-resolution counting mode at a pixel size of 0. Images were recorded using SerialEM A total of 24, micrographs were collected between two datasets recorded on the same microscope. The resulting approximately , particles from each dataset were refined using non-uniform refinement 63 , and combined before being subjected to further classification and refinement. Particles were refined locally using a mask including the TMD and the fiducial, followed by local refinement with a mask only including the TMD. The resulting 3. After successive rounds of RosettaCM, the model was locally fit using Coot 0. The model was refined in real space using Phenix 1. Protein was then mixed to a final protein concentration of 2. Counts were then measured using a Microbeta2 scintillation counter in 96 well plates with triplicate measurements Mutants were evaluated similarly from cell lysates of transfected cells. Data were fit to a single-site binding equation using Graphpad Prism. Competition binding experiments were performed at the same protein concentration in the same detergent buffer. Measurements were done in triplicates and fit with a one-site competitive binding equation in Graphpad Prism. After 10 min, cells were spun down and resuspended in the same buffer containing 2. Transport was stopped by the addition of ice-cold buffer, and the cells were collected on Glass Fiber C filters. The filters were then counted in scintillation fluid. Finally, the unconstrained protein was subjected to ns NPT simulations. Periodic boundary conditions were employed for all simulations, and the particle mesh Ewald PME method 81 was used for long-range electrostatic interactions with the pair list distance of The simulation time step was set to 2 fs with the covalent hydrogen bonds constrained with the SHAKE algorithm Langevin dynamics was applied with a piston period of 50 fs and a piston decay of 25 fs as, well as Langevin temperature coupling with a friction coefficient of 1 ps - 1. Three independent runs of ns were performed for each system, denoted as run1, run2, and run3. Snapshots from trajectories were recorded every ps. Molecular structures of protonated dopamine and serotonin were adopted from the previous studies 84 , The exhaustiveness of the simulation was set to 50, and the algorithm returned a maximum of 20 ligand binding poses. MD trajectory analysis was performed in VMD The yellow trace is the fluorescence of mVenus and the black trace is of Trp. Residues in EL1 that are not resolved in the cryo-EM map are also noted. A representative micrograph defocus —1. The workflow depicts the data processing scheme used to reconstruct VMAT2. Two datasets were collected comprising 7, and 17, micrographs respectively. Movies were corrected for drift using patch motion correction and resultant micrographs were used to estimate defocus and pick particles. Blob picking followed by template picking was utilized to select approximately 5 million particles from each dataset. This resulted in approximately k particles after combining both datasets. Particles underwent non-uniform refinement and further rounds of 2D classification and heterogeneous refinement to select particles with higher resolution features. Local refinements with a mask that excluded the detergent micelle further improved the resolution of the reconstruction. Bayesian polishing was utilized to correct local particle motion followed by further rounds of 2D classification, heterogeneous refinements, and CTF refinement. The final stack of 92k particles was then subjected to local refinement to produce the final unsharpened map. DeepEMhancer was used to locally sharpen the map for interpretation. The dotted line indicates an FSC value of 0. The ligand conformations are shown in cyan sticks with blue stick illustrating cryo-EM resolved binding pose. The variations of W are displayed in purple sticks with dark purple showing the cryo-EM-resolved orientation. Docking simulations identified e, the most favorable —9. The positions of mutated residues are shown boxed and in red. The positions of human variants are shown in blue boxes. Alphafold grey. Dopamine and serotonin are displayed in violet and cyan as van der waals VDW surfaces. The amine group of dopamine and serotonin is in close contact with E and their respective hydroxyl group s interact with R This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Views, downloads and citations are aggregated across all versions of this paper published by eLife. Significance of findings important : Findings that have theoretical or practical implications beyond a single subfield landmark fundamental important valuable useful. Strength of evidence solid : Methods, data and analyses broadly support the claims with only minor weaknesses exceptional compelling convincing solid incomplete inadequate. Abstract Summary The vesicular monoamine transporter 2 VMAT2 is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Introduction Neuronal signaling by monoaminergic neurotransmitters controls all aspects of human autonomic functions and behavior, and dysregulation of this leads to many neuropsychiatric diseases. Figure 1. Figure 2. VMAT2 conformation and residues involved in gating. Figure 3. Tetrabenazine recognition and binding. Figure 4. Mechanism of tetrabenazine inhibition, gating mechanisms, and polar networks. Extended Data Figure 1. Biochemical characterization, construct design, and sequence conservation of VMAT2. Extended Data Figure 2. Extended Data Figure 3. Extended Data Figure 4. Tetrabenazine docking and molecular dynamics simulations. Extended Data Figure 5. Point mutants in tetrabenazine binding site. Extended Data Figure 6. Extended Data Figure 7. Docking-predicted binding poses of dopamine and serotonin. Sign up for email alerts Privacy notice.

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