Structural aspects of calcium-release activated calcium channel function

Abstract

Store-operated calcium (Ca(2+)) entry is the process by which molecules located on the endo/sarcoplasmic reticulum (ER/SR) respond to decreased luminal Ca(2+) levels by signaling Ca(2+) release activated Ca(2+) channels (CRAC) channels to open on the plasma membrane (PM). This activation of PM CRAC channels provides a sustained cytosolic Ca(2+) elevation associated with myriad physiological processes. The identities of the molecules which mediate SOCE include stromal interaction molecules (STIMs), functioning as the ER/SR luminal Ca(2+) sensors, and Orai proteins, forming the PM CRAC channels. This review examines the current available high-resolution structural information on these CRAC molecular components with particular focus on the solution structures of the luminal STIM Ca(2+) sensing domains, the crystal structures of cytosolic STIM fragments, a closed Orai hexameric crystal structure and a structure of an Orai1 N-terminal fragment in complex with calmodulin. The accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.

Keywords: Orai channel proteins; calcium release activated calcium; calmodulin; store operated calcium entry; stromal interaction molecules.

Figure 1. Domain architecture of human STIM and Orai proteins. (A) STIM1 and STIM2 domain composition. STIM proteins are targeted to the SR/ER through signal sequences (S; purple). STIM proteins contain a canonical Ca²⁺ binding EF-hand (cEF; yellow), non-canonical EF-hand (nEF; red) and SAM domain in the lumen (green). A single pass TM (purple) section separates the luminal domains from the cytosolic CC domains (CC1, CC2, CC3; cyan). Pro/Ser (P/S; orange) and poly-Lys-rich (poly-K; blue) regions are found in the non-conserved cytosolic segments of the proteins. (B) Orai1, Orai2 and Orai3 domain composition. The cytosolic N-terminal domain (NTD), extracellular loop1 (EL1), cytosolic loop 2 (CL2), extracellular loop3 (EL3) and cytosolic C-terminal domain (CTD) are colored beige while the transmembrane TM1, TM2, TM3 and TM4 domains are purple. In (A and B), residue ranges defining the domain boundaries are indicated above the linear depiction.

Figure 2. Ca²⁺-loaded STIM1 and STIM2 EF-SAM structural features. (A) Compact α-helical fold of Ca²⁺-loaded STIM1 EF-SAM. The 10 α-helices making up the compact fold of EF-SAM are labeled. (B) The STIM1 EF-hand hydrophobic cleft composition. The hydrophobic amino acids which create the non-polar cavity are shown as sticks (purple) on the surface depiction of the EF-hand domain. Residues which do not contribute to the cleft, but align with hydrophobic components of the STIM2 EF-hand cleft are colored cyan. (C) The STIM1 SAM domain hydrophobic anchors which pack into the hydrophobic cleft. The SAM domain is shown as a surface representation with hydrophobic anchor residues depicted as sticks (red). Residues which do not contribute to the hydrophobic core, but align with residues that are buried non-polar components of the STIM2 SAM domain are colored cyan. (D) Compact α-helical fold of Ca²⁺-loaded STIM2 EF-SAM. The 10 α-helices making up the compact fold are labeled. (E) The STIM2 EF-hand hydrophobic cleft composition. The hydrophobic residues which create the non-polar cavity are shown as sticks (purple and cyan) on the surface representation of the EF-hand domain. (F) The STIM2 SAM domain is shown as surface representation with hydrophobic anchor resides depicted as sticks (red). Residues which contribute to the hydrophobic core of the STIM2 SAM, but are not structurally conserved in the STIM1 SAM domain are shown in cyan. In (A–F), the domain color is consistent with Figure 1 and Ca²⁺ is shown as orange spheres; N and C denote amino and carboxy termini, respectively. Images were created with 2K60.pdb and 2L5Y.pdb coordinates for STIM1 and STIM2 EF-SAM, respectively.^,

Figure 3. Human and C. elegans structures of the cytosolic CAD/SOAR/ccb9 domains. (A) Crystal structure of a Leu374Met/Val419Ala/Cys436Thr triple mutant human CAD/SOAR/ccb9 dimer. The 4 α-helices making each monomer are labeled. The residues which were mutated to stabilize the dimer and facilitate crystallization are shown as sticks (blue). The region of intramolecular supercoiling between CC2 and CC3 helices are shaded teal. The intermolecular angle between the CC2 helices at the Tyr361 pivot point is indicated. (B) Crystal structure of the C. elegans CC1-CAD/SOAR/ccb9 dimer. The 2 α-helices making up each monomer are labeled. The CC1 helix demonstrated to modulate STIM1 activation is shaded magenta. Unresolved regions of low electron density are shown as broken black lines. In (A and B), the amino and carboxy termini are denoted by N and C, respectively. The human and C. elegans structure images were created with 3TEQ.pdb and 3TER.pdb coordinates, respectively.

Figure 4.D. melanogaster Orai channel structure. (A) Cytosolic view of the Orai hexamer structure in the presumably closed state. An individual dimer unit building block is bounded by a broken black box. The TM1, TM2, TM3 and TM4 segments from a single monomer are labeled. (B) Residue composition of the TM1-consituted pore region. Only 2 TM1 segments exhibiting the greatest separation are shown for clarity. The acidic (red), hydrophobic (green), and basic (blue) pore-lining residues are shown relative to the extracellular space and the cytosol. The residue position mutated in a heritable form of severe combined immunodeficiency disease (i.e., R91W in human numbering) is labeled as ‘SCID’. The direction of the Ca²⁺ gradient (i.e., high to low concentration) is indicated with an arrow. (C) The TM4 C-terminal extension within the dimer unit. The antiparallel interaction between the C-terminal extensions is shown with hydrophobic residues involved in stabilizing this dimer interface depicted as sticks (green). The hinge regions responsible for creating the antiparallel orientation of the C-terminal extensions are indicated. In (A–C), color is consistent with Figure 2, and the amino and carboxy termini are labeled N and C, respectively. The D. melanogaster structure images were created with the 4HKR.pdb coordinate file.

Figure 5. Rat Ca²⁺-CaM structure in complex with a human Orai1 N-terminal fragment. (A) Dumbbell structure of Ca²⁺-loaded CaM forming a complex with a fragment of the Orai1 N-terminal domain (i.e., residues 69–91) through interactions at the C-lobe. The locations of the CaM lobes, central linker helix between lobes and the Orai1 N-terminal helix (beige) are labeled. (B) The Ca²⁺-CaM C-lobe hydrophobic cleft. The residues constituting the hydrophobic cleft are depicted as purple sticks. The anchor residues from the Orai1 N-terminal fragment which pack into the CaM C-lobe hydrophobic cavity are shown as red sticks. In (A and B), the amino and carboxy termini are labeled N and C, respectively, and the Ca²⁺ atoms are shown as orange spheres. The complex structure images were created with the 4EHQ.pdb coordinate file. All structure images in Figures 1–5 were rendered using The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.