Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation

Abstract

Pentameric ligand-gated ion channels mediate fast chemical transmission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric ligand-gated ion channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway.X-ray crystallography | allostery | signal transduction | cys-loop receptor P entameric ligand-gated ion channels (pLGICs) are a superfamily of membrane receptors that mediate fast chemical transmission of nerve signals in the central and peripheral nervous system (1). These allosteric receptors couple neurotransmitter (agonist) binding in the extracellular domain (ECD) to the opening of the ionic pore located in the transmembrane domain (TMD). There are two classes in pLGIC, with either cationic channels (acetylcholine -nAChR -and 5HT3 receptors) or anionic ones (glycine and GABA receptors). The molecular understanding of their allosteric transitions is a central issue in the pharmacology of pLGICs, as it would allow the rational design of novel orthosteric and allosteric ligands. Recently reported full-length structures of several prokaryotic and eukaryotic members of the family have provided significant insights into the conserved architecture of these receptors (2-5). Nevertheless, little is known about the structural events that link the binding/unbinding of agonist to the opening/closure of the channel gate. To understand the gating mechanism, the structures of the same receptor in different allosteric states are needed at atomic resolution. Here we report two crystal structures of wildtype GLIC, a proton-gated bacterial ion channel from Gloeobacter violaceus (6) at two different pHs, above and below pH 50 .GLIC structure wa...

Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein

“…Accordingly, the blooming quaternary structure is evident for all three members of the 2-aminopyrimidine series and emphasizes the importance of the conformational differences in quaternary structure associated with the state changes. These sequence patterns of quaternary rearrangements reflected in distances between diametric subunits are in close correspondence in residue positions with those found in the open and closed channel forms of GLIC (13).…”

“…Data for 32 and 33 overlay very well and do not differ much from 15 across the entire sequence, especially for the abovementioned regions with greatest torsional motions that are not observed for nicotine. However, the patterns of dihedral angle changes are far smaller than seen in the GLIC structure and AChR models (13,26,27). …”

“…Nicotine also adds Y89 to the perturbed side chains. Changes in quaternary structure noted in reference to the gating transition of prokaryotic GLIC, as blooming (13,27), are not seen for nicotine and carbamylcholine, where the Apo form of the receptor is used as a reference state. However, it should be recognized that the apo-receptor contains Hepes buffer occupying the reference site (16).…”

“…Values were compared with those obtained for the nicotine complex and the open and closed states of prokaryotic GLIC protein (13). For the α-carbon distances (Fig.…”

“…An exception to this is the α7-neuronal nAChR composed of five identical subunits and exhibiting a high degree of cooperativity for agonist activation (9). Recently, sequence alignments identified genes coding for pentameric ligand-gated ion channels in prokaryotes led to the resolution of the first structure by X-ray crystallography on 3D crystals of a pentameric receptor protein from Erminia chrysanthemi (ELIC) (10) and Gloeobacter violaceus (GLIC) (11,12) and provided high-resolution structures of the two end point states of the cooperative gating mechanism in the same pentameric ligand-gated ion channel (GLIC) (13). Recently, the first structure of a eukaryotic member of the family, the anionic glutamate receptor from Caenorhabditis elegans (GluCl), was solved at atomic resolution (14), revealing remarkable identity of 3D structure with GLIC.…”