In the last few years, there has been a critical shift in the cryo-electron microscopy field, bringing near-atomic resolution structural analysis of small protein complexes and integral membrane proteins into the realm of possibility for the first time. This year, we have applied these new technologies to several membrane proteins of interest. Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. In 2015, we showed that when AMPA receptors transition to the active state, there is 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 GluK2 showed how these changes accommodate channel closing. We have continued these studies and are now close to a higher resolution structure, making it potentially the first instance of high-resolution structure determination of a glutamate receptor in its desensitized state. In addition to the iGluRs, we have also taken on structural studies of the ion channel CorA, in collaboration with Eduardo Perozo from the University of Illinois. The 200 kDa pentameric membrane channel CorA is the major Mg2+ uptake system in bacteria. CorA contributes to Mg2+ homeostasis through a negative feedback loop, where Mg2+ binding at the subunit interface leads to channel closure and low Mg2+ concentrations stabilize the open conformation. Several crystal structures have shed light on the architecture of the magnesium-bound closed state, while electron paramagnetic resonance (EPR) spectroscopic studies of purified CorA revealed large quaternary conformational changes associated with magnesium binding/unbinding. Our 3.8 Angstrom structure of the 5-fold symmetric Mg2+ bound CorA is similar to structures obtained by X-ray crystallography. However, in the absence of Mg2+, our analyses reveal at least two distinct, asymmetric conformations of the CorA channel, determined to 7 Angstrom resolution. In these structures, the interfaces between subunits are lost, with the subunits splaying out, and resulting in an open pore. Our findings suggest a novel mechanism for channel opening, whereby sequential loss of Mg2+ ions at low Mg2+ concentration leads to destabilization of the 5-fold symmetric state, which, in turn allows movement of the intracellular domains and opening of the transmembrane region. We also undertook a study of human P-glycoprotein, a small, pseudosymmetric member of the ABC transporter family that is responsible for the export of a variety of small molecule compounds. While we were unable to achieve high resolution with our structures of this very small, highly flexible integral membrane protein, our analysis did reveal a conformational landscape for the ATPase cycle of this protein. We found that, unexpectedly, the protein samples both the open (nucleotide binding domains separated) and closed (nucleotide binding domains close together) conformations in the apo and ATP-bound states. In the post-hydrolysis state, we found that the protein is locked in a single, closed conformation; after release of phosphate, and before exchange of ADP for ATP, P-glycoprotein samples a continuum of open conformations. While we continue efforts to obtain higher resolution, the closed conformation discovered in our present work already represent a novel structure that does not match any existing crystal structures for a member of the ABC transporter family.
| 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 |
| 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 |
| Frank, Gabriel A; Shukla, Suneet; Rao, Prashant et al. (2016) Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle. Mol Pharmacol 90:35-41 |
| 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 |
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