CryoEM Structure of a Prokaryotic Cyclic Nucleotide-gated Ion Channel

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2017 Apr 12

PMID 28396445

Citations 42

Authors

Zachary M James

Andrew J Borst

Yoni Haitin

Brandon Frenz

Frank DiMaio

William N Zagotta

David Veesler

Affiliations

Soon will be listed here.

Abstract

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from -which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

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References

Ludtke S, Baldwin P, Chiu W . EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol. 1999; 128(1):82-97. DOI: 10.1006/jsbi.1999.4174. View

Lee C, MacKinnon R . Structures of the Human HCN1 Hyperpolarization-Activated Channel. Cell. 2017; 168(1-2):111-120.e11. PMC: 5496774. DOI: 10.1016/j.cell.2016.12.023. View

Emsley P, Lohkamp B, Scott W, Cowtan K . Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 4):486-501. PMC: 2852313. DOI: 10.1107/S0907444910007493. View

Zagotta W, Olivier N, Black K, Young E, Olson R, Gouaux E . Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature. 2003; 425(6954):200-5. DOI: 10.1038/nature01922. View

Wainger B, DeGennaro M, Santoro B, Siegelbaum S, Tibbs G . Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature. 2001; 411(6839):805-10. DOI: 10.1038/35081088. View

Mindell J, Grigorieff N . Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol. 2003; 142(3):334-47. DOI: 10.1016/s1047-8477(03)00069-8. View

Yang H, Hu M, Guo J, Ou X, Cai T, Liu Z . Pore architecture of TRIC channels and insights into their gating mechanism. Nature. 2016; 538(7626):537-541. DOI: 10.1038/nature19767. View

Veesler D, Cupelli K, Burger M, Graber P, Stehle T, Johnson J . Single-particle EM reveals plasticity of interactions between the adenovirus penton base and integrin αVβ3. Proc Natl Acad Sci U S A. 2014; 111(24):8815-9. PMC: 4066496. DOI: 10.1073/pnas.1404575111. View

Gordon S, Zagotta W . Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels. Neuron. 1995; 14(4):857-64. DOI: 10.1016/0896-6273(95)90229-5. View

10.

Saponaro A, Pauleta S, Cantini F, Matzapetakis M, Hammann C, Donadoni C . Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function. Proc Natl Acad Sci U S A. 2014; 111(40):14577-82. PMC: 4210022. DOI: 10.1073/pnas.1410389111. View

11.

Brelidze T, Carlson A, Sankaran B, Zagotta W . Structure of the carboxy-terminal region of a KCNH channel. Nature. 2012; 481(7382):530-3. PMC: 3267858. DOI: 10.1038/nature10735. View

12.

Chen V, Arendall 3rd W, Headd J, Keedy D, Immormino R, Kapral G . MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1):12-21. PMC: 2803126. DOI: 10.1107/S0907444909042073. View

13.

Durr K, Chen L, Stein R, De Zorzi R, Folea I, Walz T . Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell. 2014; 158(4):778-792. PMC: 4263325. DOI: 10.1016/j.cell.2014.07.023. View

14.

Scheres S . RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol. 2012; 180(3):519-30. PMC: 3690530. DOI: 10.1016/j.jsb.2012.09.006. View

15.

DiMaio F, Song Y, Li X, Brunner M, Xu C, Conticello V . Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat Methods. 2015; 12(4):361-365. PMC: 4382417. DOI: 10.1038/nmeth.3286. View

16.

Li X, Mooney P, Zheng S, Booth C, Braunfeld M, Gubbens S . Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods. 2013; 10(6):584-90. PMC: 3684049. DOI: 10.1038/nmeth.2472. View

17.

Lolicato M, Nardini M, Gazzarrini S, Moller S, Bertinetti D, Herberg F . Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem. 2011; 286(52):44811-20. PMC: 3247997. DOI: 10.1074/jbc.M111.297606. View

18.

Roseman A . FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. J Struct Biol. 2004; 145(1-2):91-9. DOI: 10.1016/j.jsb.2003.11.007. View

19.

Kimanius D, Forsberg B, Scheres S, Lindahl E . Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife. 2016; 5. PMC: 5310839. DOI: 10.7554/eLife.18722. View

20.

Song Y, DiMaio F, Wang R, Kim D, Miles C, Brunette T . High-resolution comparative modeling with RosettaCM. Structure. 2013; 21(10):1735-42. PMC: 3811137. DOI: 10.1016/j.str.2013.08.005. View