We have made significant progress towards using 3D cryo-electron microscopy to determine the structures of membrane proteins and ligand-induced conformational changes at resolutions of 20 Angstrom or better. Our work has included structures of numerous membrane protein assemblies, including chemotaxis receptors, multi-drug transporters and ion channels. We are currently using single-particle analysis of solubilized membrane proteins to obtain high resolution structures of these types of molecules, exemplified by our work on glutamate receptors. Ionotropic glutamate receptors (iGluRs) are major mediators of excitatory synaptic transmission in the central nervous system and play a vital role in mediating memory and learning. The AMPA, kainate and NMDA receptor subtypes work by opening a cation-selective pore in response to ligand binding, a key step in intercellular communication in the nervous system. Channel opening is followed by receptor desensitization that closes the channel, with both sets of reactions occurring on a millisecond time scale. High resolution crystallographic studies of isolated amino terminal domain (ATD) and ligand binding domain (LBD) dimers, coupled with decades of biochemical and functional studies, have provided important insights into the structure and function of these receptor components, while crystallographic analysis of the closed state of a modified form of the AMPA receptor (GluA2cryst) has enabled delineation of domain organization and transmembrane (TM) structure in the context of the tetrameric receptor assembly in an antagonist-bound closed state. However, no structures of either active or desensitized conformations have been reported. Given that a number of earlier studies provide hints of extensive conformational variability in closed, active, and desensitized states, including the ability of subunits to move independently during the activation process, it seems likely that trapping functionally relevant states of native receptors in the context of 3D crystals may be challenging. Our previous structural studies of a full-length kainate receptor (GluK2) at 20 Angstrom resolution using cryo-electron tomography and sub-volume averaging suggested that desensitization involves dramatic structural changes in the LBD, with minimal changes in the ATD. These findings are in contrast to the extensive quaternary rearrangements of ATD tetramers observed in earlier single particle negative stain analyses on AMPA receptors at 40 Angstrom resolution. However, neither of these analyses was at resolutions high enough to provide a molecular interpretation of the domain movements involved. To determine how glutamate receptor ion channels accommodate the structural changes necessary for activation and desensitization, we carried out single particle cryo-electron microscopy (cryo-EM) of both the AMPA receptor GluA2 and the kainate receptor GluK2. To better understand how structural changes gate ion flux across the membrane, we trapped AMPA and kainate receptor subtypes in their major functional states and analyzed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a corkscrew motion of the receptor assembly, driven by closure of the ligand binding domain. Desensitization is accompanied by rupture of the amino terminal domain tetramer in AMPA, but not kainate receptors, with a 2-fold to 4-fold symmetry transition in the ligand binding domains in both subtypes. Our 7.6 Angstrom structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical, and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. Our findings thus provide a detailed glimpse into the overall gating cycle of glutamate receptors, an evaluation of the similarities and differences in conformational changes observed in AMPA and kainate receptor families, and a molecular mechanism for the dramatic movements of the LBD that occur during the receptor gating cycle. A central value of single particle cryo-EM methods we have used is that they enable definition of these functionally important large scale structural changes without the potential constraints introduced by disulfide cross links or crystal lattice contacts that may restrict conformational changes in 3D crystals used for crystallographic analyses. At the same time, the need to use detergents or amphipols to stabilize membrane proteins for structural analysis has the potential to disrupt functionally important protein-lipid interactions. Whether this impacts ligand gated ion channel structures is an important area for future research. Other challenging problems include obtaining a structural understanding of how glutamate receptor activation, at lower agonist concentrations than used in the present study, leads to sub-conductance states. In addition, it will be important to explore central mechanistic questions such as how individual LBDs move independently during the activation process, how gating occurs in heteromeric iGluR assemblies such as NMDA receptors, and whether the extent of cleft closure, which varies for partial agonists, has any consequence on either open state or desensitized state structures.
| Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram (2018) Microbiology catches the cryo-EM bug. Curr Opin Microbiol 43:199-207 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558:553-558 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Publisher Correction: Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature : |
| Earl, Lesley A; Falconieri, Veronica; Milne, Jacqueline Ls et al. (2017) Cryo-EM: beyond the microscope. Curr Opin Struct Biol 46:71-78 |
| Wohlbold, Teddy John; Podolsky, Kira A; Chromikova, Veronika et al. (2017) Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2:1415-1424 |
| Meyerson, Joel R; Chittori, Sagar; Merk, Alan et al. (2016) Structural basis of kainate subtype glutamate receptor desensitization. Nature 537:567-571 |
| Subramaniam, Sriram; Earl, Lesley A; Falconieri, Veronica et al. (2016) Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41:194-202 |
| Stuart, David I; Subramaniam, Sriram; Abrescia, Nicola G A (2016) The democratization of cryo-EM. Nat Methods 13:607-8 |
| Matthies, Doreen; Dalmas, Olivier; Borgnia, Mario J et al. (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating. Cell 164:747-56 |
| Subramaniam, Sriram; Kühlbrandt, Werner; Henderson, Richard (2016) CryoEM at IUCrJ: a new era. IUCrJ 3:3-7 |
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