Basak S et al. (2020), High-resolution structures of multiple 5-HT3AR-...

XB-ART-58092

Elife 2020 Oct 16;9. doi: 10.7554/eLife.57870.

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High-resolution structures of multiple 5-HT3AR-setron complexes reveal a novel mechanism of competitive inhibition.

Basak S , Kumar A , Ramsey S , Gibbs E , Kapoor A , Filizola M , Chakrapani S .

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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. Molecular dynamic simulations of these structures embedded in a fully-hydrated lipid environment assessed the stability of ligand-binding poses and drug-target interactions over time. Together with simulation results of apo- and serotonin-bound 5-HT3AR, the study reveals a distinct interaction fingerprint between the various setrons and binding-pocket residues that may underlie their diverse affinities. In addition, varying degrees of conformational change in the setron-5-HT3AR structures, throughout the channel and particularly along the channel activation pathway, suggests a novel mechanism of competitive inhibition.

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Species referenced: Xenopus laevis
GO keywords: serotonin binding [+]

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	Figure 1 with 2 supplements Cryo-EM structure of 5-HT3AR-setron complexes. (a) Three-dimensional reconstruction of 5-HT3AR-Alo at 2.92 Å resolution (left) and the corresponding structural model (right) that shows the overall architecture consisting of the extracellular domain (ECD), transmembrane domain (TMD), and structural regions of the intracellular domain (ICD). The alosetron density is shown in deep olive color and the three sets of glycans are shown as stick representation. Arrow points toward the setron density. Solid line denotes putative membrane limits. (b) Extracellular view of 5-HT3AR-Alo (left), 5-HT3AR-Ondan (middle), and 5-HT3AR-Palono (right) maps sliced at the neurotransmitter-binding site. In each case, the five molecules of respective setrons are highlighted in colors. Chemical structures of setrons are shown above.
	Figure 2 with 2 supplements Setron-binding poses. (a) Cryo-EM density for the setrons, located at the canonical neurotransmitter-binding site. The map is contoured at 9σ (5-HT3AR-Grani) (Basak et al., 2019); 8.5σ (5-HT3AR-Palono); 7σ (5-HT3AR-Ondan); 6σ (5-HT3AR-Alo). The binding site lies at the interface of the principal (colored) and the complementary (gray) subunits. The binding-site residues are shown in stick representation with residues from the principal subunit labeled in black and those from the complementary subunit in magenta. From top to bottom: 5-HT3AR-Grani, 5-HT3AR-Palono, 5-HT3AR-Ondan, and 5-HT3AR-Alo. (b) LigPlot analysis of setron-5-HT3AR interactions. Most interactions with the setron are hydrophobic in nature (shown by red arch with spikes). Putative hydrogen bond between Trp156 and alosetron is shown as a green dotted line.
	Figure 3 with 2 supplements Assessment of conformation stability of ligand-binding poses by molecular dynamic simulations. (a) Time evolution of root mean square deviation (RMSD) of setrons’ and serotonin’s heavy atoms relative to their initial cryo-EM conformations of 5-HT3AR for each protomer subunit. (b) Representative views of various palonosetron orientations during the 100 ns simulation. When the tertiary amine nitrogen in the bicyclic ring is pointing up, it interacts with the carbonyl oxygen of Trp156 and when it points down, it interacts with carbonyl oxygen of Asn101 side chain. (c) Time evolution of Root Mean Square Deviation (RMSD) of the bicyclic ring to its initial cryo-EM position.
	Figure 4 with 1 supplement Setron-binding pocket and conformational changes in Loop C. (a) Global alignment of 5-HT3AR-Apo, 5-HT3AR-State1 (serotonin-bound), 5-HT3AR-Grani, 5-HT3AR-Palono, 5-HT3AR-Ondan, and 5-HT3AR-Alo structures. With respect to 5-HT3AR-Apo, the serotonin- and setron- bound conformations reveal an inward positioning of Loop C (shown by arrow). (b) Relative displacement of the inner β-strands seen from a side-view (left panel) and the outer β-strands seen from the top (right panel). Arrows indicate the direction of movement. (c) Pentameric assembly of setron- and serotonin-bound structures were aligned to Apo-5-HT3AR. A cubic spline interpolation was then done to smoothly connect cα displacement for each structure and mapped by short cylinders, whose diameters are equivalent to the displacement at that position compared to Apo-5-HT3AR. The color was also scaled to the same value using the color map shown. The analysis was done in Matlab v2019a (Mathworks, Natick MA).
	Figure 5 Assessment of the number of water molecules present within each ligand-binding site during MD simulation. (a) Snapshots during the simulation showing water molecules in the pocket. (b) Average number of water molecules (defined as a count of water oxygen atoms within 3 Å of any setron atoms) for each setron- and serotonin-bound simulation subdivided by protomer and the corresponding standard deviation.
	Figure 6 Dynamic interaction between Arg65 and Asp202. (a) Time evolution of the minimum distance between side-chain polar atoms of Arg65 and Asp202 throughout 100 ns simulations. A 4 Å distance threshold is shown as a red dashed line to denote a generous cutoff for H-bond interactions between these residues. (b) MD snapshot that show the Arg65-Asp202 interaction. (c) Dose-response curve for serotonin activation measured by TEVC recordings (at −60 mV) for WT 5-HT3AR and R65A expressed in oocytes. The EC50, the Hill coefficient (nH), and the number of independent oocyte experiments are: WT (EC50: 2.70 ± 0.09 μM; nH: 2.3 ± 0.17; n: 3) and R65A (EC50: 13.79 ± 0.50 μM; nH: 4.4 ± 0.59; n: 4) (Basak et al., 2019) (d) Functional analysis of Arg65. Currents were elicited in response to serotonin (concentrations used near EC50 values WT- 2 μM, and R65A- 10 μM) with and without co-application of setrons. Dotted arrows show the extent of setron inhibition in each case. (e) A plot of the ratio of peak current in the presence of setron to peak current in the absence of setron is shown for WT and R65A. Data are shown as mean ± s.d (n is indicated in parenthesis). Significance at p=0.01 (*) and p=0.05 () calculated by two sample t-test for wild type and R65A.
	Figure 7 with 2 supplements Pore profiles of 5-HT3AR in Apo, serotonin-, and setron- bound states. (a) Ion conduction pathway predicted by HOLE (Smart et al., 1996). Models are shown in cartoon representation. Only two subunits are shown for clarity. The locations of pore constrictions are shown as sticks. (b) The pore radius is plotted as a function of distance along the pore axis. The dotted line indicates the approximate radius of a hydrated Na+ ion which is estimated at 2.76 Å (right) (Marcus, 1988).

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Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed