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.

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-Ca ²⁺ concentration and directly binds to the store-operated Ca ²⁺ channel Orai1 for its activation when Ca ²⁺ recedes. At high resting ER-Ca ²⁺ 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 Ca ²⁺ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca ²⁺ entry via Orai1, decreasing the Ca ²⁺ oscillation frequency as well as store-operated Ca ²⁺ 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.

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca²⁺ levels ([Ca²⁺]_i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca²⁺]_i through store-operated Ca²⁺ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca²⁺]_ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca²⁺ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins.

Calcium (Ca ²⁺ ) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca ²⁺ signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani , affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani -infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca ²⁺ sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca ²⁺ , and the Ca ²⁺ -binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca ²⁺ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca ²⁺ and undergo minimal secondary and tertiary structural changes upon Ca ²⁺ -binding, in contrast to several Ca ²⁺ -binding proteins. Of note, SlCBL1-2 binds Ca ²⁺ with apparent K _D values of 526 ± 113 µM and 28.7 ± 9.47 µM, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca ²⁺ -binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca ²⁺ sensor in defense signalling.