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.

The Orai Pore Opening Mechanism

“…Main differences were detected within the pore region and the helical segments at the outmost side of the channel complex. Consistently, the pore region of all open structures exhibits a dilation of the basic region of approximately 10 Å ( Figure 2 I,J) compared to 6 Å in the closed structures [ 34 , 35 , 36 , 51 , 71 ]. The most drastic structural differences occur within the channel periphery.…”

“…These extracellular loop1 regions were described to shape structured turrets which are stabilized via a network of hydrophobic interactions. They establish an electronegative pore entrance [ 51 ] matching with the findings that the three glutamates in each loop1 function as a CAR [ 45 ].…”

“…Among the currently available structures, the two closed-state structures include dOrai wildtype and a LoF mutant (dOrai K163W) ( Figure 2 A,D), while the open-state structures contain single-point mutations known to induce constitutive activity (H206A (in Orai1 H134A) or P288L (in Orai1 P245L)) ( Figure 2 B,C,E) independent of STIM1 [ 34 , 35 , 36 , 51 ]. Among the open-state structures, only the recent cryo-EM structure of dOrai H206A shows a high resolution with 3.3 Å [ 51 ].…”

“…In the closed state, the C-termini of adjacent subunits cross each other in an antiparallel manner and form a belt-like structure around the channel due to coiled-coil interactions ( Figure 2 A). In the open crystal structures, the entire TM4 C-terminus segment is fully straightened toward the cytosol via unlatching conformational changes (dOrai H206A and P288L) ( Figure 2 B,C) [ 34 , 51 ]. Contrarily, the cryo-EM open (dOrai P288L) structure shows neither the antiparallel pairing nor a straightening of the TM4 helices, but rather a tight packing of TM3/TM4 ( Figure 2 E–J) [ 34 ].…”

“…Recent studies published four main crystal and two cryo-EM (cryogenic electron microscopy) structures of Drosophila melanogaster Orai (dOrai) [ 34 , 35 , 36 , 51 ], which are discussed in more detail later in the review. These structures consistently revealed a hexameric stoichiometry of the Orai channel and a single pore-forming region in the center of the complex.…”