Our application of emerging tools in cryo-EM to the study of membrane proteins and their conformational changes continues to grow. Progress on two projects is summarized below. G protein-coupled receptors (GPCRs) comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways through coupling with downstream signaling mediators, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestins. The structural mechanisms that define how GPCRs selectively couple to a specific type of G protein or arrestin remain unknown. Using cryo-EM, we have now shown in a paper published in the journal Nature in June 2018, that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi alpha-subunit, which is wedged into the cytoplasmic face of the transmembrane helix bundle and directly contacts the N-terminus of Helix-8 of rhodopsin. Comparison of the structures of inactive, Gi- and arrestin- bound forms of rhodopsin with inactive and Gs- bound forms of the beta-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with Gs, Gi, and arrestin recognition by activated GPCRs. Voltage-activated potassium (Kv) channels open to conduct K+ ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. In a paper published in August 2018 in the journal eLife, we report the structure of the Kv1.2-2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy (cryo-EM). At a resolution of 3 Angstrom for the cytosolic domain and 4 Angstrom for the transmembrane domain, the structure determined in nanodiscs using cryo-EM is remarkably similar to the previously determined X-ray structure. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.
| 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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