Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution toward the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signaling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. Based on these findings we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

The structural basis for the association of eukaryotic and prokaryotic protein receptors and their triple-helical collagen ligand remains poorly understood. Here, we present the crystal structures of a high affinity subsegment of the Staphylococcus aureus collagen-binding CNA as an apoprotein and in complex with a synthetic collagen-like triple helical peptide. The apo-protein structure is composed of two subdomains (N1 and N2), each adopting a variant IgG-fold, and a long linker that connects N1 and N2. The structure is stabilized by hydrophobic interdomain interactions and by the N2 C-terminal extension that complements a b-sheet on N1. In the ligand complex, the collagen-like peptide penetrates through a spherical hole formed by the two subdomains and the N1-N2 linker. Based on these two structures we propose a dynamic, multistep binding model, called the 'Collagen Hug' that is uniquely designed to allow multidomain collagen binding proteins to bind their extended rope-like ligand.

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signaling1–4. Store operated Ca2+ entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially-restricted biotin-labelling in situ coupled with mass spectrometry5, 6 and a secondary screen based on bimolecular fluorescence complementation7, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches lead to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca2+-NFAT signaling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.