Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Structural Plasticity Allows Calumenin-1 to Moonlight as a Ca2+-Independent Chaperone: Pb2+ Enables Probing Alternate Inhibitory Conformation.

Human calumenin-1 (HsCalu-1) is an endoplasmic reticulum (ER) and Golgi-resident Ca2+-binding protein of the hepta-EF-hand superfamily that plays a vital role in maintaining the cytoplasmic Ca2+ concentration below toxic levels by interacting with Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and ryanodine receptors (RyR), indicating its role in Ca2+ homeostasis in the ER. HsCalu-1 seems to be able to exhibit structural plasticity to achieve its plethora of functions. In this study, we demonstrate that HsCalu-1 acts as a chaperone in both its intrinsically disordered state (apo form) and the structured state (Ca2+-bound form). HsCalu-1 chaperone activity is independent of Ca2+ and Pb2+ binding attenuating its chaperone-like activity. Incidentally, Pb2+ binds to HsCalu-1 with lower affinity (KD = 38.46 µM) (compared to Ca2+-binding), leading to the formation of a less-stable conformation as observed by a sharp drop in its melting temperature Tm from 67 °C in the Ca2+-bound form to 43 °C in the presence of Pb2+. The binding site for Pb2+ was mapped as being in the EF-Hand-234 domain of HsCalu-1, a region that overlaps with the Ca2+-dependent initiator of its functional fold. A change in the secondary and tertiary structure, leading to a less-stable but compact conformation upon Pb2+ binding, is the mechanism by which the chaperone-like activity of HsCalu-1 is diminished. Our results not only demonstrate the chaperone activity by a protein in its disordered state but also explain, using Pb2+ as a probe, that the multiple functions of calumenin are due to its ability to adopt a quasi-stable conformation.

The Differential Response to Ca2+ from Vertebrate and Invertebrate Calumenin Is Governed by a Single Amino Acid Residue.

Calumenin (Calu) is a well-conserved multi-EF-hand-containing Ca2+-binding protein. In this work, we focused on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologues with respect to its response to Ca2+ binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca2+ free form and responds to Ca2+ with a dramatic gain in structure. Calumenin from Caenorhabditis elegans (CeCalu) is structured even in the apo form, with no conformational change upon binding of Ca2+. We decode this structural and functional distinction by identifying a single "Leu" residue-based switch located in the fourth EF-hand of HsCalu1, occupied by "Gly" in the invertebrate homologues. We demonstrate that replacing Leu with Gly (L150G) in HsCalu1 enables the protein to adopt a structural fold even in the Ca2+ free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca2+). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly or Leu). Our analyses reveal that the Leu that replaced Gly from fishes onward is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca2+-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue strongly corroborates the change in the structure of the protein and its pleiotropic functions and seems like it can be extended to the presence or absence of a heart in that organism.

STIM Proteins and Regulation of SOCE in ER-PM Junctions.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.