Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The seven gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits, which assemble to form three major functional families named after the ligands that were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate, and NMDA. Given their essential role in normal brain function and development and increasing evidence that dysfunction of iGluR activity mediates several neurological and psychiatric diseases as well as damage during stroke, we devote substantial effort to analyzing GluR function at the molecular level. Atomic resolution structures solved by protein crystallization and x-ray diffraction provide a framework within which to design electrophysiological and biochemical experiments to define the allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. The resulting information will allow the development of subtype-selective antagonists and allosteric modulators with novel therapeutic applications while revealing the inner workings of a complicated protein machine that plays a major role in brain function.
Crystallographic analysis of glutamate receptor–ligand complexes
Mayer; in collaboration with Jane
The recent crystallization of the ligand-binding cores of AMPA, kainate, and NMDA receptor subunits, and a related bacterial receptor from the photosynthetic bacterium Synechocystis sp. PCC 6803, which we named GluR0, has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists, providing insight into the mechanisms of activation and desensitization. During the past year, experimental efforts in structural biology have focused on studies of the ligand-binding cores of members of the kainate receptor gene family and on studies of the cytoplasmic domains of NMDA receptors.
Although a large number of glutamate receptor agonist and partial agonist structures have been solved, relatively few antagonist complexes have been crystallized. This is likely the result of the different conformations of the closed-cleft agonist-bound structure and open-cleft antagonist-bound structure and the differences in mobility of the ligand protein complex. Agonist-bound complexes crystallize easily and have similar temperature factors in domains 1 and 2; antagonist-bound structures are hard to crystallize. High-resolution structures of the ligand-binding cores bound to two novel GluR5-selective antagonists, UBP302 and UBP310, reveal a hyperextended conformation in which the ligand forces the domains to separate to a greater extent than observed in the GluR2 apo structure. Strikingly, electron density was excellent for domain 1 but less well defined for domain 2. Refinement of the final structure required TLS (translation/libration/screw) analysis and revealed both domain breathing motions and higher overall mobility of domain 2. The structure of the UBP302 and 310 complexes is strikingly different from those previously solved for the GluR2 DNQX and NR1 5,7DCKA complexes and revealed a mode of ligand binding that could not be modeled from earlier structural information. We expect that the new information will facilitate the design of new subtype kainate receptor antagonists.
Mayer ML. Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity. Neuron 2005;45:539-552.
Crystallographic and functional analysis of kainate receptor dimer interfaces
Ghoshal, Mayer; in collaboration with Rosenmund, Schuck
Despite overall high amino acid–sequence homology and similar gating kinetics, the exchange of single amino acids that differ between the AMPA and kainate receptor subtypes of iGluRs leads to the identification of a nondesensitizing AMPA receptor L483Y construct that, during the past five years, has found wide application as a tool to investigate numerous aspects of iGluR biology. The availability of similar constructs for kainate receptors would be of great interest, yet surprisingly paradoxical results have been obtained with the introduction of mutations into the ligand-binding core/dimer interface of kainate receptors. Accordingly, we crystallized the GluR5 ligand-binding core in a dimeric assembly similar to that observed for AMPA receptors and determined its structure at 2.1 Å resolution. Using the structure as a template, we designed a series of mutants in the dimer interface with the goal of stabilizing the active conformation. We crystallized the mutants and solved their structures and then examined the sedimentation behavior of the purified ligand-binding cores by analytic ultracentrifugation. We predicted that the mutations that stabilized kainate receptor dimer formation would attenuate desensitization. Using HEK293 cell patches, we tested our prediction by undertaking rapid perfusion experiments, which revealed a complete block of GluR6 desensitization for constructs that stabilize the dimer assembly. Of interest for another construct, which was designed to reproduce the local dimer structure of the GluR2 L483Y mutant, we observed no block of desensitization or change in sedimentation behavior, even though, when crystallized, the resulting structure closely resembled the L483Y parent. Such results illustrate the importance of using a wide range of biophysical approaches to characterize receptor function, given that crystal structures cannot currently be used to estimate reliably the energetics of assembly of macromolecular complexes, even though they capture the molecular fine details of protein assembly.
Horning MS, Mayer ML. Regulation of AMPA receptor gating by ligand binding core dimers. Neuron 2004;41:379-388.
Mayer ML. Glutamate receptor ion channels. Curr Opin Neurobiol 2005;15:282-288.
Mayer ML. Some assembly required. Nat Struct Mol Biol 2005;12:208-209.
COLLABORATORS
David Jane, PhD, University of Bristol, Bristol, UK
Christian Rosenmund, PhD, Baylor College of Medicine, Houston, TX
Peter Schuck, PhD, Division of Bioengineering and Physical Sciences, ORS, NIH, Bethesda, MD
For further information, contact mlm@helix.nih.gov.