Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation
Abstract
The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou, Burstein, & Long, 2018). Here, we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-EM. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.
Data availability
The cryo-EM map and the atomic model have been deposited in the Electron Microscopy Data Bank (EMDB) and in the Protein Data Bank (PDB) with accession numbers EMD-23002 and 7KR5, respectively.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R01GM094273)
- Stephen Barstow Long
National Institute of General Medical Sciences (R35GM131921)
- Stephen Barstow Long
National Cancer Institute (P30CA008748)
- Stephen Barstow Long
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Hou et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,983
- views
-
- 598
- downloads
-
- 40
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Both immunoglobulin light-chain (LC) amyloidosis (AL) and multiple myeloma (MM) share the overproduction of a clonal LC. However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic-soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered the understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a2 conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.