Abstract
Serotonin receptors (5-HT3AR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron. Each structure reveals a distinct interaction fingerprint between the setron and binding-pocket residues that may underlie their diverse affinities. In addition, setrons elicit varying degrees of conformational change throughout the channel that, quite surprisingly, lie along the channel activation pathway, suggesting a novel mechanism of competitive inhibition. Molecular dynamic simulations were used to assess binding-poses and the drug-target interaction dynamics. Together, this study provides a molecular basis for setron binding affinities and their inhibitory effects.
Introduction
Cancer treatments by radiation or chemotherapy triggers the release of excess serotonin from the mucosal enterochromaffin cells in the upper gastrointestinal tract1. Serotonin binds to serotonin (3) receptors (5-HT3Rs), a pentameric ligand-gated ion channel (pLGIC), on the vagal afferent nerve in the gut and on the chemoreceptor trigger zone in the brainstem leading to severe nausea and vomiting in the patients. These common side effects of cancer treatment take a significant physical and psychological toll on cancer patients. Without management, these side effects can reduce patient compliance, undermining treatment success2. Furthermore, uncontrolled debilitating side-effects result in secondary complications such as dehydration and anorexia that require additional hospitalization and increase overall healthcare costs.
Current antiemetic therapies include a 5-HT3R antagonist treatment regimen, which is considered a major advancement in improving patient quality of life during cancer treatment. Setrons, competitive antagonists of 5-HT3R, are effective in the prevention of chemotherapy-induced nausea and vomiting (CINV), radiation therapy-induced nausea and vomiting (RINV), and postoperative nausea and vomiting (PONV) 3,4. CINV nausea occurs in acute and delayed phases. The first generation of FDA approved setrons are effective for treating acute but not delayed phase nausea due to their short plasma half-lives. They belong to the following major classes based on their chemical structures: carbazole (e.g. ondansetron), indazole (e.g. granisetron), indole (e.g. dolasetron, tropisetron), and imidazole (e.g. alosetron). Although setrons share the same fundamental mechanism of action, they have varying efficacies, dose-response profiles, duration of action, and off-target responses. These differences perhaps underlie variable patient response, particularly in the context of acute and refractory emesis5. Palonosetron, the only FDA approved second generation setron, a derivative of isoquinoline, is shown to have a longer half-life, improved bioavailability, and efficacy. In addition, palonosetron is implicated in causing receptor internalization, which further improves anti-emetic properties. Beyond their role in controlling emesis, setrons are used to treat GI disorders including irritable bowel syndrome (IBS), obesity, and several inflammatory, neurological and psychiatric disorders such as migraine, drug abuse, schizophrenia, depression, anxiety, and cognitive disorders. However, in some cases, toxicity and adverse side-effects have hampered their use. For example, with alosetron, which was approved by the FDA for the treatment of diarrhea-predominant IBS, led to severe ischemic colitis in many patients6. Given the broad therapeutic potential of 5-HT3AR antagonists, the prospect of substantial therapeutic gains by probing the setron pharmacophore is encouraging, as well as developing novel pharmaceuticals with higher efficacy and reduced side-effects.
At the physiological level, the 5-HT3Rs play an important role in gut motility, visceral sensation, and secretion 7-12, and are also implicated in pain perception, mood, and appetite. 5-HT3Rs are the only ion channels13 among the large family of serotonin receptors, the rest being GPCRs. 5-HT3Rs are expressed as homopentamers of subunit A or heteropentamers of subunit A, in combination with B, C, D, or E subunits14. Compositional and stoichiometric differences lead to differential responses to serotonin, gating kinetics, permeability, and pharmacology15-17. This functional diversity, tissue specific expression patterns, and distinct pathophysiology of 5-HT3R isoforms establish a need for subtype specific drugs to address diverse clinical needs18. Of note, granisetron, ondansetron, palonosetron, and alosetron have slightly different affinities for various receptor subtypes19. Ondansetron, in addition to binding to 5-HT3Rs, also binds to several GPCRs, such as 5-HT1BR, 5-HT1CR, α1-adrenergic receptors and μ-opioid receptors20. Granisetron binds to all subtypes of 5-HT3R, but has little or no affinity for 5-HT1R, 5-HT2R and 5-HT4R receptors. Palonosetron is highly selective for 5-HT3AR and dolasetron for 5-HT3BR21. Structural insights into setron-binding poses came initially from crystal structures of the acetylcholine binding protein (AChBP), bound to granisetron, tropisetron, or palonosetron22-24 and more recently from 5-HT3AR complexed with tropisetron and granisetron25,26. While some of the basic principles of setron-binding are now clear, there is still limited understanding of differing pharmacodynamics among setrons and the associated clinical relevance.
In the present study, we have solved cryo-EM structures of the full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron at the resolution range of 2.9 Å to 3.3 Å. Together with our previously solved structure of 5-HT3AR in complex with granisetron (grani-5-HT3AR), we provide details of various setron-binding modes and the ensuing conformational changes that lead to channel inhibition. Using molecular dynamics (MD) simulations and electrophysiology, we have further validated setron-binding modes and interactions within the conserved binding pocket. Combined with abundant functional, biochemical, and clinical data, these new findings may serve as a structural blueprint of drug-target interactions that can guide new drug development.
Results
Cryo-EM structures of setron-5-HT3AR complexes
Structures of the full-length 5-HT3AR in complex with setrons were solved by single-particle cryo-EM. Detergent solubilized 5-HT3AR was incubated with 100 µM of palonosetron, ondansetron, or alosetron for 1 hour prior to vitrification on cryo-EM grids. Iterative classifications and refinement produced a final three-dimensional reconstruction at a nominal resolution of 3.3 Å for 5-HT3AR-Palono (with 91,163 particles), 3.0 Å for 5-HT3AR-Ondan (67,333 particles), and 2.9 Å for 5-HT3AR-Alo (46,065 particles) (Supplemental Figures 1a and 1b). The local resolution of the map was estimated using ResMap and in the range of 2.5-3.5 Å for each of these reconstructions (Supplemental Figure 1c). Structural models were built using refined maps containing density for the entire extracellular domain (ECD), transmembrane domain (TMD), and the structured regions of the intracellular domain (ICD) (Fig. 1a and Supplemental Figure 2). Overall, each of setron-bound 5-HT3AR complexes has an architecture similar to previously solved 5-HT3A receptors25,27-29. Among the non-protein densities present in the map at the ECD are the three sets of peripheral protrusions corresponding to N-linked glycans (Fig. 1a, right) and a strong, unambiguous density at each of the intersubunit interfaces, corresponding to individual setrons (Figs. 1b). Besides this site, no additional densities for setrons were found under these conditions, although there have been predictions that palonosetron may act as both an orthosteric and allosteric ligand30.
The map quality was particularly good at the ligand-binding site allowing us to model sidechains and the setron orientation. Setrons bind within the canonical neurotransmitter binding pocket and are lined by residues from Loops A, B, and C on the principal (+) subunit and Loops D, E, and F from the complementary (-) subunit (Fig. 2). Residues within 4 Å of setron include Asn101 in Loop A, Trp156 in Loop B, Phe199 and Tyr207 in Loop C, Trp63 and Arg65 in Loop D, and Tyr126 in Loop E. These residues are strictly conserved, and perturbations at each of these positions impact efficacy of setrons and serotonin31-33. In each setron-5-HT3AR complex, the essential pharmacophore of setron is placed in a similar orientation: the basic amine is at the deep-end of the pocket in the principal subunit; the defining aromatic moiety interacts with residues in the complementary subunit; and the carbonyl-based linker, between the two groups, is essentially coplanar with the aromatic ring.
The basic amine of the setron is in a bicyclic ring in granisetron and palonosetron, and a diazole ring in ondansetron and alosetron. The amine is within 4 Å of Trp156 (loop B), Tyr207 (loop C), Trp63 (loop D) and Tyr126 (loop E), and are likely to be involved in polar interactions with these residues. In particular, the carbonyl oxygen of Trp156 is close to the amine group of setron, and in the 5-HT3AR-Alo, it forms a hydrogen bond with the amine group in the diazole ring. The relative orientation of the tertiary nitrogen and Trp156 is conducive for a cation-pi interaction, as seen in the AChBP-5-HT3 chimera structure22. A similar interaction is also predicted for the primary amine group of serotonin34. The aromatic, hydrophobic end of the molecule is an indazole in granisetron, isoquinoline in palonosetron, carbazole in ondansetron, and imidazole in alosetron. It is oriented toward the complementary subunit, and lies parallel to the membrane. In this orientation, the aromatic moiety is stabilized by a number of hydrophobic interactions with Ile44, Trp63, Tyr64, Ile201, and Tyr126. The setron molecule is within 4-5 Å and potentially makes π-π interactions (edge-to-face or face-to-face) with Trp63, Tyr126, Trp156, and Tyr207. These interactions are also consistent with our MD simulations (discussed below). In addition, the planar aromatic rings lie beneath Arg65, and in close proximity to the positively charged nitrogen in the guanidinium group of Arg65, revealing a potential cation-pi interaction (Fig. 3a). This interaction was also observed in the AChBP-5-HT3 chimera22 and 5-HT3AR-Grani structures26. As previously noted in 5-HT3AR-Grani, the setron position causes reorientation of Arg65 (β2 strand or loop D) and Trp168 (β8-β9; loop F)26. Earlier reports also predicted large orientational differences for Trp168 when the binding-site was occupied by agonist or antagonist35. In this position, Arg65 is in a network of interactions involving Asp42 (β1), Try126 (β6), Trp168 (β8-β9; loop F), Arg169 (β8-β9; loop F), and Asp177 (β8-β9; loop F) (Fig. 3b). Glu102 (loop A) which is in the vicinity of ligand binding site is in a hydrogen bond network with Thr133 and Ala134 carbonyl (β6 strand). Interestingly, both of these networks are also present in serotonin-bound 5-HT3AR, but absent in 5-HT3AR-Apo, indicating the ligand-induced formation of the interaction network28,29.
Our initial expectation was that, as a highly-potent competitive antagonist, setrons would stabilize a 5-HT3AR-Apo like conformation. However, 5-HT3AR-Grani revealed a counter-clockwise twist of beta strands in the ECD leading to a small inward movement of Loop C (connecting β9-β10 strands) closing-in on granisetron. The Loop C conformation has been correlated to the nature of the ligand in the binding site and agonist efficacy. The AChBP-ligand complexes have shown that agonist binding induces a “closure” of Loop C, capping the ligand-binding site36. This conformational change may be part of a conserved pLGIC mechanism that couples ligand binding to channel opening through the ECD-TMD interfacial loops. Antagonist-bound structures show Loop C further extended outward36, while partial agonists seem to induce partial Loop C closure but not to the level achieved by agonists23. The 5-HT3AR and other pLGIC structures solved thus far, in the apo and agonist-bound states, follow this general trend 25,28,29. However, studies have shown that unliganded pLGIC gating kinetics remain unaffected by Loop C truncation37, raising ambiguity over its role in the channel opening mechanism. In comparison to the 5-HT3AR-Apo, the Loop C conformation in the 5-HT3AR-setron structures are positioned inward to a varying degree, and in the 5-HT3AR-Alo the orientation is similar to the serotonin-bound conformation (State-I)26 (Fig. 3c). The twisting inward movement does not pertain to Loop C alone, it was also seen in adjoining β7, β9 and β10 strands that form the outer-sheets of the β-sandwich core, with notable deviation from 5-HT3AR-Apo in the vicinity of the binding pocket (Fig. 3d, right panel). There are minimal changes in the β-strands of the inner sheets (β1, β2, β6) (Fig. 3d, left panel; Supplemental Figure 3). These conformational changes approach those seen in serotonin bound 5-HT3AR (State-I)29.
Conformational changes are also present in the TMD and may arise from small twisting movements in the ECD. Interestingly, in each of the 5-HT3AR-setrons structures, the pore-lining M2 helices are positioned away from the central axis, and are in a more-expanded conformation than the 5-HT3AR-Apo (Fig. 4a). At positions Val260 (13′), Leu260 (9′), Ser 253 (2′), and Glu250 (−1′), where the pore is constricted to below the hydrated Na+ radii38 in 5-HT3AR-Apo, the pore-radii is larger in 5-HT3AR-setron structures (Fig. 4b). However, these conformations are expected to remain non-conducting because Leu260 (9′) is constricted to ∼2.3 Å. While there are small conformational changes in the ICD, the post-M3 loop occludes the lateral portals at the interface of the TMD and ICD which are ion exit paths. The extent of occlusion is similar to what was seen in 5-HT3AR-apo28.
Molecular Dynamics Simulations
Several structural analyses of 5-HT3AR-palono, 5-HT3AR-ondan, and 5-HT3AR-alo were performed by investigating 100 ns MD simulations of these complexes embedded in a 1-palmitoyl-2-oleoyl phosphatidyl choline (POPC) membrane and encased in water with 150 mM NaCl. To assess the stability of each setron binding-pose modeled from cryo-EM density, we quantified the root mean square deviation (RMSD) of each pose relative to its starting conformation, averaged across each subunit. We found that all setrons maintained a low RMSD (<2.5 Å), with palonosetron demonstrating the largest RMSD among the group (Fig 5a). During the simulation, the bicylic ring displayed considerable fluctuation and positional reorientation. In these positions, palonosetron had distinct interactions with binding site residues (Supplemental Figure 4). To evaluate the types of interactions that these setrons maintained with protein sidechains during MD simulation, we calculated 5-HT3AR-setron interaction fingerprints (see figure legend or methods for full interaction type definitions) for each protomer in the complex (Supplemental Figures 5 and 6). Hydrophobic interactions comprise the majority of these interaction fingerprints, with some contributions from water-mediated interactions and aromatic interactions. Notably, the alosetron fingerprints suggests that this compound forms stronger interactions with Asp202 and Trp156 when compared to palonosetron, ondansetron, and granisetron.
To characterize the flexibility of Loop C in our 5-HT3AR-setron simulations, we evaluated a number of structural quantities. First, we assessed the RMSD of Loop C for each protomer in each 5-HT3AR-setron simulation by evaluating the distances of Cα, carbonyl carbon, and backbone nitrogen atoms of residues Ser200 through Asn205 referenced to their initial cryo-EM conformations (Supplemental Figure 7a). These data suggest that Loop C is stable in its initial cryo-EM conformation, particularly in the 5-HT3AR-Alo and 5-HT3AR-Ondan simulations, but can also adopt an alternate ‘open’ conformation where Loop C extends away from the binding site surface. To further evaluate Loop C conformational flexibility in our MD simulations we defined a custom dihedral formed by the Cα atoms of residues Ala208, Phe199, Glu198, and Ile203 that measured the orientation of the loop relative to the principal protomer binding site (Supplemental Figure 7b). This dihedral was defined in such a way that a large angle would denote that the loop is oriented away from the binding site and small or negative angles indicate that the loop is oriented towards the binding site. This data demonstrates that Loop C tends to remain in its initial cryo-EM resolved conformation, particularly in the 5-HT3AR-Alo MD simulation.
To assess the impact of Loop C movement on the relative size of each setron-binding pocket, we quantified the number of water molecules found within each binding site. This was evaluated by counting water oxygen atoms within 3 Å of any setron atom. Since each setron remained stable within its respective binding site, this measurement represents an approximation of binding-site volume. This data shows that alosetron and ondansetron have a lower number of water molecules within their binding sites (Fig 5b and 5c).
To understand the motion of Loop C between the ‘closed’ cryo-EM structure and the MD sampled ‘open’ conformation we evaluated the minimum polar side chain atom distance between Arg65 and Asp202, residues known to form a hydrogen bond interaction that may effectively rigidify loop C in a ‘closed’ conformation39. We hypothesized that in our MD simulations Loop C would not adopt an ‘open’ conformation if an Arg65-Asp202 interaction was formed. We find that the 5-HT3AR-Alo MD simulation maintained an interaction between Arg65 and Asp202 more often than in any other setron-bound structure, and that most setron-bound simulations did not appreciably form this stabilizing interaction (Fig 6a and 6b). Thus, our mechanistic hypothesis is such that when Arg65 is interacting with Asp202, Loop C is in a stable ‘closed’ conformation, which in turn reduces the accessibility of the binding pocket to water, and incidentally contributes to the higher stability of the ligand binding pose.
Our MD simulations predict that Arg65 may have a differential effect on the binding of various setrons. In agreement, mutations at the Arg65 position in human 5-HT3AR abolish granisetron binding but tropisetron binding is only reduced40. To further assess the role of Arg65 in binding various setrons, we measured the extent of inhibition of serotonin-induced currents. Since for competitive antagonists, the extent of inhibition depends on agonist concentration, the serotonin concentration in each case was kept close to the EC50 value for wild-type (2 μM) and R64A (10 μM) (Fig 6c). Granisetron and palonosetron inhibition was measured at 1 nM; ondansetron and alosetron inhibition was measured at 0.1 nM (these concentrations where chosen to achieve a 50% inhibition for wild type upon co-application) (Fig 6d and 6e). Of note, co-application of setron in some cases has ∼100 fold lower effect than pre-application due to slow on-rates41. Mutational perturbation at Arg65 has a significant effect on inhibition by each setron, albeit to varying extents.
Conclusion
A comprehensive structural analysis of multiple high-resolution structures of setron-bound 5-HT3AR complexes reveal several features of competitive antagonism that were not fully evident from the previous structural findings. Serotonin binds within a partially solvent-exposed cavity at the subunit interface and elicits Loop C closure, which may be coupled to channel opening. The setron-binding pocket, while involving overlapping residues, extends further into the complementary subunit. Setron-binding evokes varying degrees of Loop C closure and in some cases, almost to the same degree as a serotonin-bound state. The Loop C movements are associated with varying degrees of structural changes in the inner and outer β-strands that translate to small changes in the pore-lining M2 helices. Overall, setrons stabilize 5-HT3AR conformational states that are non-conductive, but appear to lie between the apo and serotonin-bound states. These findings therefore suggest that competitive antagonism in 5-HT3AR, and potentially in other pLGIC, may involve stabilizing intermediates along the activation pathway. With new emerging uses of setrons to treat psychiatric disorders, inflammation, substance abuse, and Alzheimer’s disease, these studies lay the foundation for the design of newer therapeutics with higher treatment efficacy and fewer off-target effects.
Methods
Electrophysiological measurements in oocytes
Mouse 5-HT3AR gene (purchased from GenScript) and mutant genes were inserted into pTLN plasmid. The plasmids were linearized with Mlu1 restriction enzyme by digesting overnight at 37 °C. The mMessage mMachine kit (Ambion) was used to make mRNA as per the manufacturer’s protocol and cleanup using RNAeasy kit (Qiagen). 3–10 ng of mRNA was injected into X. laevis oocytes (stages V–VI), and incubated for 2-5 days, after which current recordings were performed. Water injected oocytes were used as a control to verify that no endogenous currents were present. Female X. laevis were purchased from Nasco and kindly provided by W. F. Boron. Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University approved the animal experimental procedures. Oocytes were maintained in OR3 medium (GIBCO-BRL Leibovitz medium containing glutamate, 500 units each of penicillin and streptomycin, pH adjusted to 7.5, osmolarity adjusted to 197 mOsm) at 18 °C. Warner Instruments Oocyte Clamp OC-725 was used to perform two-electrode voltage-clamp experiments at a holding potential of −60 mV. Currents were sampled and digitized at 500 Hz with a Digidata 1332A. Clampfit 10.2 (Molecular Devices) was used to analyze experimental data. Perfusion solution consisted of 96mM NaCl, 2mM KCl, 1.8mM CaCl2, 1mM MgCl2, and 5mM HEPES (pH 7.4, osmolarity adjusted to 195 mOsM) was used at a flow rate of 6 ml/min. Chemical reagents (serotonin hydrochloride, alosetron hydrochloride, ondansetron hydrochloride, and palonosetron hydrochloride) were purchased from Sigma-Aldrich.
Full-length 5-HT3AR cloning and transfection
The mouse 5-HT3AR (NCBI Reference Sequence: NM_001099644.1) gene was codon-optimized for Spodoptera frugiperda (Sf9) cells and purchased from GenScript. The construct consists of the 5-HT3AR gene along with a C-terminal 1D4-tag42 and four strep-tags (WSHPQFEK) at the N terminus, each separated by a linker sequence (GGGSGGGSGGGS) and followed by a TEV-cleavage sequence (ENLYFQG). Sf9 cells (Expression System) were grown in ESF921 medium (Expression Systems) at 28 °C without CO2 exchange and in absence of antibiotics. Cellfectin II reagent (Invitrogen) was used for transfection of recombinant 5-HT3AR bacmid DNA into sub-confluent Sf9 cells. After 72 h of transfection, the progeny 1 (P1) recombinant baculoviruses were obtained by collecting the cell culture supernatant. The P1 was then used to infect Sf9 cells which produced P2 viruses, and subsequently P3 viruses from the P2 virus stock. The P3 viruses were used for recombinant protein expression.
5-HT3AR expression and purification
Sf9 cells are grown to approximately 2.5 × 106 per ml followed by infection with P3 viruses. After 72 h post-infection, the cells were centrifuged at 8,000g for 20 min at 4 °C to separate the supernatant from the pellet. The cell pellet was resuspended in 20 mM Tris-HCl, pH 7.5, 36.5 mM sucrose supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). Cells were sonicated on ice. Non-lysed cells were pelleted down by centrifugation (3,000g for 15 min) and the supernatant was collected. The membrane fraction was separated by ultracentrifugation (167,000g for 1 h) and solubilized in 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 0.5% protease inhibitor and 1% C12E9 for 2 h at 4 °C. Non-solubilized material was removed by ultracentrifugation (167,000 g for 15 min). The solubilized membrane proteins containing 5-HT3A receptors were bound with 1D4 beads pre-equilibrated with 20 mM HEPES pH 8.0, 150 mM NaCl and 0.01% C12E9 for 2 h at 4 °C. The non-bound proteins were removed by washing beads with 100 column volumes of 20 mM HEPES pH 8.0, 150 mM NaCl, and 0.01% C12E9 (buffer A). The protein was then eluted with 3 mg/ml 1D4 peptide (TETSQVAPA) which is solubilized in buffer A. Eluted protein was deglycosylated with PNGase F (NEB) by incubating 5 units of the enzyme per 1 μg of protein for 2 h at 37 °C under gentle agitation. Deglycosylated protein was then purified using a Superose 6 column (GE healthcare) equilibrated with buffer A. Purified protein was concentrated to 2–3 mg/ml using 50-kDa MWCO Millipore filters (Amicon) for cryo-EM studies.
Cryo-EM sample preparation and data acquisition
5-HT3AR protein (∼2.5 mg/ml) was filtered and incubated with 100 μM drugs (Alosetron, Ondansetron, and Palonosetron) for 1 hour. Fluorinated Fos-choline-8 (Anatrace) was added to the protein sample to a final concentration of 3 mM. The protein was then blotted onto Cu 300 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools) two times with 3.5 μl sample each time, and the grids were plunge frozen immediately into liquid ethane using a Vitrobot (FEI). The grids were imaged using a 300 kV FEI Titan Krios G3i microscope equipped with a Gatan K3 direct electron detector camera. Movies containing ∼50 frames were collected at 105,000× magnification (set on microscope) in super-resolution mode with a physical pixel size of 0.848 Å/pixel, dose per frame 1 e-/Å2. Defocus values of the images ranged from −1.0 to −2.5 µm (input range setting for data collection) as per the automated imaging software SerialEM43.
Image processing
MotionCor44 was used to correct beam-induced motion using a B-factor of 150 pixels2. Super-resolution images were binned (2×2) in Fourier space, making a final pixel size of 0.848 Å. Entire data processing was conducted in RELION 3.145. CTF of the motion-corrected micrographs were estimated using Gctf software46. Auto-picked particles from total micrographs (Table 1) from individual datasets (each drug) were subjected to 2D classification to remove suboptimal particles. An initial 3D reference model was generated from the 5-HT3AR-apo cryo-EM structure (RCSB Protein Data Bank code (PDB ID): 6BE1). The model was low-pass filtered at 60 Å using EMAN247. Iterative 3D classifications, 3D auto-refinements, and bayesian polishing generated density model of Alosetron, Ondansetron and Palonosetron bound 5-HT3AR with 42, 065 particles, 67, 333 particles, and 91,163 particles, respectively. Per-particle contrast transfer function (CTF) refinement and beam tilt correction were applied followed by a final 3D-autorefinement. A soft mask was generated in RELION and used during the post-processing step, which resulted in an overall resolution of 2.92 Å, 3.06 Å, and 3.32 Å for 5-HT3AR-Alo, 5-HT3AR-Ondan and 5-HT3AR-Palono, respectively (calculated based on the gold-standard Fourier shell coefficient (FSC) = 0.143 criterion, Table 1). B-factor estimation and map sharpening were performed in the post-processing step in RELION. The ResMap program was used to calculate local resolutions 48.
5-HT3AR model building
The final refined models have clear density of residues Thr7–Leu335 and Leu397–Ser462. The unobserved density at the region of (336–396) is comprised of an unstructured loop which links the amphipathic MX helix and the MA helix. The 5-HT3AR-apo cryo-EM structure (PDB ID: 6BE1) was used as an initial model and refined against its EM-derived map using PHENIX software package 49, using rigid body, local grid, NCS, and gradient minimization parameters. COOT is used for manual model building 50. Real space refinement in PHENIX yielded the final model with a final model to map cross-correlation coefficient of 0.834 (5-HT3AR-Palono), 0.846 (HT3AR-Ondan), and 0.848 (5-HT3AR-Alo). Stereochemical properties of the model were validated by Molprobity51. The pore profile was calculated using the HOLE program52. Figures were prepared using PyMOL v.2.0.4 (Schrödinger, LLC).
MD Simulation Setup and Protocol
The cryo-EM-derived structures of palonosetron-bound, alosetron-bound, and ondansetron-bound 5-HT3AR pentamer were prepared for MD simulations with the Protein Prep Wizard in the SchrÖdinger scientific software suite 2019-2 using default settings (Small-Molecule Drug Discovery Suite 2019-2, Schrödinger, LLC, New York, NY, 2019). This protocol adds missing hydrogen atoms to the initial protein-ligand complex. After the initial preparatory steps and protonation assignment of side chains, a brief restrained energy minimization in vacuo using the OPLS3 force field53 was carried out to finalize system setup for each protein-ligand complex. Each setron-5-HT3AR complex was then embedded into a 1-palmitoyl-2-oleoyl phosphatidyl choline (POPC) bilayer using the Membrane Builder tool of the CHARMM-GUI webserver 54. The system was then solvated with TIP3P water and 150 mM NaCl were added to the simulation system by replacing random water molecules. Excess sodium ions were added to neutralize the charge of each protein-ligand complex. The resulting simulation systems had initial dimensions of ∼130×130×207 Å3 and consisted of the 5-HT3AR pentamer, the setrons bound to each 5-HT3AR subunit, ∼400 POPC molecules, ∼83,000 water molecules, ∼240 sodium ions, and ∼220 chloride ions, for a total of ∼330,000-346,000 atoms. Throughout this work we reference data from our previously published simulation of granisetron-bound 5-HT3AR26 in comparison to simulations of these three novel setron-bound 5-HT3AR complexes.
The CHARMM36m forcefield55 was used to parameterize the protein and lipid atoms within each simulation system. Initial parameters for palonosetron, alosetron, and ondansetron were obtained from the ParamChem webserver using the CHARMM general force field 56 (https://cgenff.parmchem.org). Parameters were validated according to the procedure described56. Said validation required quantum calculations performed with Gaussian 16 (Gaussian 16, Revision C.01, Gaussian, Inc., Wallingford CT, 2016) to finalize the charges and dihedrals defined within our setron molecule models.
MD simulations were run using GROMACS 2018.657software with a timestep of 2 fs, following a steepest descent energy minimization run for 5000 steps, as well as 100 ps isothermal-isovolumetric (NVT) and 52 ns isothermal-isobaric (NPT) equilibration runs. The NVT equilibration was performed to initially heat the model systems after the steepest descents minimization. This step was performed with restraints on protein, membrane, and ligand molecule heavy atoms relative to their starting conformation. The NPT equilibration runs were performed in 5 steps of 10 ns each, within which the system was allowed to relax with gradually released restraints until finally the system was allowed to equilibrate for 2 ns of unrestrained NPT equilibration. This was followed by a 100 ns production run in isothermal-isobaric conditions. System temperature and pressure were maintained at 300 K and 1 bar, respectively, using velocity rescale58 for temperature coupling and Parrinello-Rahman barostat for pressure coupling during equilibration. Semi-isotropic pressure coupling and the Nose-Hoover thermostat59 were applied during production runs. All bonds involving hydrogens were constrained using the LINCS algorithm60. Short-range nonbonded interactions were cut at 12 Å. Long-range electrostatic interactions were computed using the Particle Mesh Ewald summation with a Fourier grid spacing of 1.2 Å.
Trajectory analyses were performed using a combination of Visual Molecular Dynamics (VMD)61 and the GROMACS analysis toolkit62 over equidistant frames of our production simulations using a 500 ps stride. In particular, all RMSD measurements and loop C orientations were obtained after aligning simulation frames onto the coordinates of the initial cryo-EM structure by comparing Cα atoms in the helices and β-sheets of the ECD. RMSD calculations were assessed for each setron by evaluating the difference in heavy atoms of the ligands between each simulation frame and the initial cryo-EM structure conformation. Similarly, Loop C RMSD’s were calculated by comparing the Cα, backbone carbonyl carbon, and backbone nitrogen atoms of residues 200 through 205 relative to their conformation in the initial cryo-EM resolved structures. To measure the orientation of Loop C, we defined a custom Loop C dihedral as being drawn between the alpha carbons of residues Ala208, Phe199, Glu198, and Ile203. To determine whether Loop C adopted a ‘closed’ or ‘open’ conformation we evaluated the distance between the Arg65 and Asp202 side chains, measured by a minimum distance of their respective polar side chain atoms for each analysed simulation frame. To evaluate how well solvated the setron binding sites were throughout our simulations, we counted the number of water oxygen atoms within 3 Å of any setron atoms for each simulation frame. Structural interaction fingerprints were calculated with an in-house python script that monitored 5-HT3AR interactions with each setron. Specifically, for each residue of 5-HT3AR, setron-protein interactions were calculated as a 9-bit representation based on the following 9 types of interactions: apolar (van der Waals), face-to-face aromatic, edge-to-face aromatic, hydrogen-bond interactions with the protein either as a donor or acceptor, electrostatic with either the protein acting as a positive or negative charge, one-water-mediated hydrogen bond, and two-water-mediated hydrogen bonds. A distance cutoff of 4.5 Å was used to identify apolar interactions between two non-polar atoms (carbon atoms), while a cutoff of 4 Å was used to evaluate aromatic and electrostatic interactions. Residue interactions were evaluated for protein side chain atoms only. Average probabilities and errors for each interaction type were estimated using a two-state Markov model, sampling the transition matrix posterior distribution using standard Dirichlet priors for the transition probabilities63.
Data Availability Accession Numbers
The coordinates of the 5-HT3AR-drugs structure and the Cryo-EM map have been deposited in wwPDB and EMDB with following accession number. PDB ID: 6W1J; EMBD ID: EMD-21511 for 5-HT3AR-Alo, PDB ID: 6W1M; EMBD ID: EMD-21512 for 5-HT3AR-Ondan and PDB ID: 6W1Y; EMBD ID: EMD-21518 for 5-HT3AR-Palono. All relevant data are available from the corresponding author upon reasonable request.
Author Contributions
S.B and S.C conceived the project and designed experimental procedures. S.B purified the protein, optimized the cryo-EM sample preparation, carried out data collection, analysis, model building, refinement, and performed two-electrode voltage-clamp recordings. A. Kumar contributed to cryo-EM data collection and functional analysis. S.C supervised the execution of the experiments, data analysis, and interpretation. E.G contributed to analysis and mapping of structural differences using MATLAB. S.R, with the assistance of A. Kapoor, performed the MD simulations, docking, and other computational analyses under the supervision of M.F. All authors contributed to manuscript preparation.
Competing Financial Interest
The authors declare that there is no competing financial interest.
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Acknowledgements
We acknowledge the use of instruments at the Cryo-Electron Microscopy Core at the CWRU School of Medicine. We are grateful to Dr. Kunpeng Li for assistance with cryo-EM imaging and data collection. We thank Denice Major for assistance with hybridoma and cell culture at the Department of Ophthalmology and Visual Sciences (supported by the National Institutes of Health Core Grant P30EY11373). We thank Dr. Walter F. Boron for kindly providing us Xenopus oocytes and for unrestricted access of the oocyte rig. We are deeply appreciative of the support provided by Dr. Fraser Moss and Mr. Brian Zeise with the oocyte rig. We are very grateful to the members of the Chakrapani lab for critical reading and comments on the manuscript. Computations were run on resources available through the Scientific Computing Facility at the Icahn School of Medicine at Mount Sinai and the Extreme Science and Engineering Discovery Environment under MCB080077 (to M.F.), which is supported by National Science Foundation grant number ACI-1548562. This work was supported by the National Institutes of Health grants R01GM108921, R01GM131216, R35GM134896, and Cryo-EM supplements: 3R01GM108921-03S1, R01GM108921-5S1, 3R01GM131216-1S1 to S.C and the AHA postdoctoral Fellowship to A.K (20POST35210394) and S.B (17POST33671152).