Discovering cryptic pocket opening and binding of a stimulant derivative in a vestibular site of the 5-HT_3Areceptor

Abstract

Ligand-gated ion channels propagate electrochemical signals in the nervous system by opening ion-selective pores in response to neurotransmitter release. A diverse set of allosteric modulators including neurosteroids, anesthetics, and lipids modulate their function in myriad ways, suggesting a complex conformational landscape. However, structures of ligand-bound ion-channel complexes can be difficult to capture by experimental techniques like cryogenic electron microscopy, particularly when binding is dynamic or transient. Here, we used computational methods to identify a possible bound state of a modulatory stimulant derivative (4-bromoamphetamine) in a cryptic vestibular pocket of a mammalian serotonin-3A receptor. Starting from an experimental activated structure containing a closed pocket, we first applied a molecular dynamics (MD) simulations-based goal-oriented adaptive sampling method to identify possible open-pocket conformations. To find plausible ligand-binding poses, we performed ensemble docking of the newly identified modulator, and reweighted docking scores by the Boltzmann energy function derived from Markov state model analysis of our trajectories. We then performed replicates of unbiased MD simulations of representative complexes in two forcefields to estimate ligand stability, and screened the most stable complexes for accessibility to the aqueous environment. For one relatively stable and accessible site, mutations predicted to disrupt lig- and binding were validated by electrophysiology recordings inXenopus laevisoocytes, and provided a mechanistic rationale for allosteric stabilization of an activated state. Given the pharmaceutical relevance of serotonin-3 receptors in emesis, pain, psychiatric and gastrointestinal diseases, characterizing relatively unexplored modulatory sites in these proteins could open valuable avenues to understanding conformational cycling and designing state-dependent drugs.

Vestibular Modulation by Stimulant Derivatives in a Pentameric Ligand-Gated Ion Channel

“…In parallel with the present study, we have observed BrAmp in particular to potentiate 5-HT 3A Rs even more potently than sTeLIC [28]. Furthermore, BrAmp modulation of 5-HT 3A Rs is sensitive to mutations at positions equivalent to sTeLIC b4-W75, b5-L93, and b6-Y104, and appears to require a similar rearrangement of the b4 residue out of the vestibular site and into contact with the complementary b5 [28]. Taken together, these findings support a general mechanism of pocket opening and ligand modulation in a subset of pLGICs including mammalian 5-HT 3A Rs.…”

“…Their work further demonstrates that covalent labeling in the vestibular site modifies 5-HT 3A R currents, supporting a functional role in receptor modulation [19]. In parallel with the present study, we have observed BrAmp in particular to potentiate 5-HT 3A Rs even more potently than sTeLIC [28]. Furthermore, BrAmp modulation of 5-HT 3A Rs is sensitive to mutations at positions equivalent to sTeLIC b4-W75, b5-L93, and b6-Y104, and appears to require a similar rearrangement of the b4 residue out of the vestibular site and into contact with the complementary b5 [28].…”

“…Indeed, multiple substitutions at residue L93 (Ala, Val, Ser, Asp) ablated functional sTeLIC expression in oocytes, consistent with a critical role for this residue in channel gating and/or assembly. In parallel work, we recently reported that mutating equivalent positions in mammalian 5-HT 3 A Rs (β4-V95, β6-N125, β5-P113) decreases sensitivity to both 5-HT activation and BrAmp potentiation (Figure 4G) [28]. This putative mechanism may be a specific feature of bacterial pLGICs and 5-HT 3 A Rs, in which the vestibular pocket exhibits conserved architecture and intersubunit interactions (Supplementary Figure S5A); in contrast, this cavity in eukaryotic receptors for GABA, glutamate, glycine, and acetylcholine are notably different and largely occluded (Supplementary Figure S5B).…”

“…F) Rotated view of superposition as in (A, inset), revealing interactions of β 4-W75 with β 5-L93 on the complementary subunit upon transitioning from closed (red) to open (green). G) View as in (F) of a mammalian 5-HT 3 A R, showing a cryo-EM structure without BrAmp (PDB ID 6DG8, orange) and a computational model with BrAmp (teal, [28]). Residues V95 and P113, corresponding to sTeLIC W75 and L93 respectively, have been shown to mediate BrAmp modulation, and are shown as sticks.…”