Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

-loaded EF-SAM (holo) contains high ␣-helicity, whereas EF-SAM in the absence of Ca 2؉ (apo) is much less compact. Accordingly, the melting temperature (T m ) of the holoform is ϳ25°C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca 2؉ -induced stabilization of 3.2 kcal mol ؊1 . We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca 2؉ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (K d ) of ϳ0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca 2؉ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca 2؉ sensor.Calcium is a fundamental signaling messenger in every eukaryotic cell, regulating a multitude of diverse and kinetically distinct cellular phenomena including gene transcription, protein folding, protein degradation, apoptosis, necrosis, and exocytosis, to name a few (1). The endoplasmic reticulum (ER) 4 is a network of folded membranes that extends through the cytoplasm to the nuclear envelope of eukaryotes. The ER membranes surround an inner cavity, the lumen, that is critical to the function of the ER as a Ca 2ϩ signaling organelle (2). Because vital Ca 2ϩ -dependent processes are associated with the ER, it is essential that changes in luminal Ca 2ϩ levels do not adversely affect these phenomena. Eukaryotes have evolved store-operated Ca 2ϩ entry (SOCE), also termed capacitive Ca 2ϩ entry, as a major Ca 2ϩ entry pathway in electrochemically non-excitable cells (3-5). SOCE is the process whereby modest ER Ca 2ϩ store depletion leads to plasma membrane (PM) Ca 2ϩ release-activated channel (CRAC) activation, providing a sustained Ca 2ϩ elevation in the cytoplasm from extracellular sources and ultimately refilling the ER luminal Ca 2ϩ stores (6). Until recently, the molecular link between ER Ca 2ϩ efflux and extracellular influx was not known. However, interfering and small inhibiting RNA studies have independently implicated stromal interaction molecule-1 (STIM1) as the likely Ca 2ϩ sensor in the ER (7,8). This single-pass, type I transmembrane protein of 685 amino acids has been found localized on both the plasma and ER membranes (3-5). The N-terminal regions of STIM1 include a signal peptide, putative EF-hand motif, and predicted sterile ␣-motif (SAM) domain. The cytosolic C-terminal region consists of two coiled-coil domains, a Pro/Ser-rich region, and a Lys-rich region (supplemental Fig. 1A) (9 -11). The putative EF-hand of STIM1 strongly aligns with the helix-loop-helix consensus sequence of this Ca 2ϩ binding motif (supplemental Fig. 1B). The EF-hand in STIM1 is somewhat unorthodox because it is seemingly unpaired, whereas Ca 2ϩ sensor pr...

Inositol 1,4,5-trisphosphate receptors (InsP3R) and ryanodine receptors (RyR) are tetrameric intracellular Ca2+ channels1. For each, the pore is formed by C-terminal transmembrane domains and regulated by signals detected by the large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224-604 of InsP3R1)2 and it requires the suppressor domain (SD, residues 1-223)2-8. We present structures of the N-terminal region (NT) of InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, the SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR19. The orientations of the three domains docked into a tetrameric structure of InsP3R10 and of the ABC domains in RyR9 are remarkably similar. The importance of the α-interface for activation of InsP3R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations9,11,12. InsP3 causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop (HS-loop) that is essential for InsP3R activation7. The loop is conserved in RyR and includes mutations associated with malignant hyperthermia and central core disease9,11,12. The HS-loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A-domain of RyR functionally replaced the SD in a full-length InsP3R, and an InsP3R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP3 and blocked by ryanodine. Activation mechanisms are conserved between InsP3R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit re-orients the first domain (SD or A-domain), allowing it, via interactions of the second domain of an adjacent subunit (IBC-β or B-domain), to gate the pore.

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca 2 binding, is found in a large number of functionally diverse Ca 2 binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca 2 binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca 2 to the open conforma-tional state in its presence. It is now clear from structures of other EF-hand proteins that this ''closed-to-open'' conformational transition is not the sole model for EF-hand protein structural response to Ca 2. More complex modes of conforma-tional change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition. Proteins 1999;37:499-507. 1999 Wiley-Liss, Inc.