Featured Arcle Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain Masahiro Enomoto 1,*, Tadateru Nishikawa 1,*, y, Sung-In Back 1, Noboru Ishiyama 1,‡, Le Zheng 1, Peter B. Stathopulos 2 and Mitsuhiko Ikura 1 1 - Princess Margaret Cancer Centre, Department of Medical Biophysics, University Health Network, University of Toronto, Toronto, ON M5G 1L7, Canada 2 - Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, London, ON, Canada N6A 5C1 Correspondence to Mitsuhiko Ikura and Peter B. Stathopulos: [email protected], [email protected] https://doi.org/10.1016/j.jmb.2019.10.003 Edited by Ichio Shimada Abstract Store operated calcium (Ca2þ) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2þ store depletion causes Orai1-composed Ca2þ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2þ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2þ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2þ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile a motif (SAM) domains (EFeSAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EFeSAM domains have been determined at high Ca2þ concentrations; however, no direct structural view of the Ca2þ binding mode has been elucidated. Further, no atomic resolution data currently exists on EFeSAM at low Ca2þ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EFeSAM binds a single Ca2þ ion with pentagonal bipyramidal geometry and an ancillary a-helix formed by the N-terminal region acts as a brace to stabilize EFeSAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary a-helix and the canonical EF-hand in low Ca2þ. Remarkably, we also detected an a-helix (þCa2þ)tob-strand (ÀCa2þ) transition at the terminal SAM domain a-helix. Collectively, our analyses indicate that one canonically bound Ca2þ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation. © 2019 Elsevier Ltd. All rights reserved. Store operated calcium (Ca2þ) entry (SOCE) is a The protein machinery that predominantly med- fundamental cell signaling process used by all iates SOCE includes the stromal interaction mole- eukaryotes. SOCE occurs when the major intracel- cule (STIM) [6e8] and the Orai proteins [9e13].The þ þ lular endoplasmic reticulum (ER) Ca2 store Orai proteins assemble as hexamers to form Ca2 - becomes depleted, which leads to opening of highly selective channels on the PM [14].TheSTIMsare Ca2þ-selective channels on the plasma membrane ER-resident proteins with functional domains (PM) that allow movement of Ca2þ down the steep located in the cytosol and ER lumen. Humans and concentration gradient from the extracellular space other vertebrates express two STIM (i.e., STIM1 into the cytosol [1e3]. Free cytosolic Ca2þ concen- and STIM2) [15] and three Orai paralogs (i.e., Orai1, trations can increase by as much as ~10-fold from Orai2, Orai3) [16]. Orai1 homomers form Ca2þ ~0.1 mMto~1mM due to SOCE, signaling a plethora release activate Ca2þ (CRAC) channels that of vital processes central to life and death and mediate a majority of the SOCE in most cell types. providing a source of Ca2þ for refilling the ER and Orai2 and Orai3 may have more tissue-specific other intracellular stores [4,5]. roles [2]. 0022-2836/© 2019 Elsevier Ltd. All rights reserved. Journal of Molecular Biology (2020) 432, 367e383 368 The Luminal STIM Region Binds One Ca2þ Ion In nonexcitable cells, SOCE can be stimulated that the EF-hand and ancillary a-helices are in downstream of any cell surface G protein-coupled conformational exchange between folded and receptor (GPCR) activation that results in inositol unfolded states. Finally, our NMR data show that 1,4,5-trisphosphate (IP3) production and IP3 recep- the C-terminal helix of the luminal SAM domain, tor channel opening on the ER, depleting ER Ca2þ which is intimately involved in maintaining the levels. However, any mode of ER Ca2þ depletion will intramolecular EF-hand:SAM domain interaction in stimulate SOCE. STIMs contain EF-hand and sterile the presence of high Ca2þ, undergoes a remarkable a-motif (SAM) domains oriented in the ER lumen and a-helix to b-strand transition in low Ca2þ. Collec- a series of coiled-coil domains in the cytosol [17,18]. tively, our crystal and solution NMR data provide These luminal and cytosolic domains have a high- previously unappreciated atomic level insights into sequence similarity among STIM orthologs [15].A the mechanisms underlying STIM activation. single pass transmembrane (TM) domain separates the luminal from the cytosolic conserved domains. STIM1 forms dimers on the ER membrane due to Results interactions by the cytosolic coiled-coil domains [19e24]. These inactive STIM1 dimers pervasively diffuse along the ER [25]. When the ER Ca2þ stores C. elegans EFeSAM is structurally similar to are full, the luminal domains remain monomeric due human STIM2 EFeSAM to Ca2þ-binding dependent stabilization of the EF- þ hand and SAM domain (EFeSAM) intramolecular In high Ca2 , the human STIM1 and STIM2 EF- interaction [26e29]. When the ER Ca2þ stores are hand and SAM domains fold into a combined depleted, this luminal intramolecular interaction is compact structure made up of ten a-helices and destabilized, leading to EFeSAM dimerization two short b-strands [27,29]. The EF-hand domain of [26,27,30e32]. This EFeSAM dimerization induces each human paralog consists of four a-helices and a marked conformational rearrangement of the two loops, whereas the SAM domain adopts a þ cytosolic coiled-coils [19,24,33] that leads to trap- compact five-helix bundle. In the Ca2 -bound ping of activated STIM1 molecules at ER-PM state, the terminal SAM helix (i.e., a10) packs into junctions [25,34]. The trapping of STIM1 occurs a hydrophobic cleft made up of the two EF-hand due to a release of a polybasic C-terminal region of motifs, thereby promoting the overall compact STIM1 that affords interactions with PM phosphoi- configuration. This conformation is further but- nositides [35e38] and due to direct interactions by tressed via the small b-sheet formed by backbone the STIM1 coiled-coils with Orai1 protein subunits hydrogen (H) bonding between loop I and II of the [39,40]. These direct STIM1eOrai1 interactions gate EF-hand motifs. the Orai1 channels. Here, our C. elegans crystal structure (1.89 Å High-resolution structures of the human STIM1 resolution) reveals the EFeSAM region of this lower and STIM2 EFeSAM have been determined by eukaryotic STIM conserves these characteristic solution NMR only in a high molar excess of Ca2þ features central to regulating the activation state of (high Ca2þ) [27,29]. However, distance restraints the molecule and SOCE (Fig. 1A). The crystal þ þ coordinating Ca2 were not directly determined in structure determined in high Ca2 shows a canoni- these structures. Furthermore, these human struc- cal helixeloopehelix motif (a1eb1ea2). Immedi- tures lacked the variable N-terminal regions shown ately downstream of the canonical EF-hand motif is a to play a modulatory role in the Ca2þ sensing second helixeloopehelix motif (a3eb2ea4), which mechanism of STIM1 and STIM2 [32,41,42]. There- pairs with the first EF-hand through backbone H- fore, the precise mechanism of Ca2þ coordination, bonding between b1 and b2, forming a short b-sheet. how the variable N-terminal region of STIMs affect The second, noncanonical EF-hand loop is not core EFeSAM structure, and mechanism of STIM clearly identifiable based on primary structure for luminal domain dimerization in buffer nominally free neither the human STIMs nor C. elegans (Fig. 1B). A of Ca2þ (low Ca2þ) remain major knowledge gaps in short linker helix (a5) links the C. elegans EF-hand the field. domain in sequence space to the five-helix bundle- Here, we present the crystal structure of the composed SAM domain (a6ea10). This SAM Caenorhabditis elegans STIM (cSTIM) luminal domain alone buries 13 residues that are at least domain in the presence of high Ca2þ, revealing a 85% solvent inaccessible (Fig. S1). single Ca2þ ion coordinated in the canonical EF- The EF-hand and SAM domains of the C. elegans hand loop with pentagonal bipyramidal geometry STIM form intimate contacts, primarily mediated by þ and that the nonconserved N-terminal region forms nonpolar interactions. In high Ca2 , these intimate a EF-hand:SAM hydrophobic interactions are a hall- an ancillary -helix that further stabilizes the core þ EFeSAM structure. In addition, we used solution mark feature of STIM Ca2 sensing that supports a NMR to probe the structure and folding of this quiescent conformation [27].InC. elegans STIM, the C. elegans luminal domain in low Ca2þ, discovering EF-hand domain forms a hydrophobic cleft made up The Luminal STIM Region Binds One Ca2þ Ion 369 of at least 13 residues with nonpolar character Ca2þ binding to the luminal domain of any STIM (Fig. 1C). This extensive hydrophobic pocket serves protein. Examination of the 2FoeFc electron density as a docking site for the hydrophobic L176 and L180 map contoured at 2.0s shows only a single location anchor residues found on the a10 helix of the SAM of high electron density outside the polypeptide domain (Fig. 1D). In C. elegans, the EF-hand:SAM chain that could be attributable to Ca2þ, despite the domain interaction is reinforced through one ionic relatively high CaCl2 concentration (i.e., 10 mM) bond involving R85 within a3 of the noncanonical used for crystallization (Fig.