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1.
Nat Chem Biol ; 17(2): 196-204, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33106661

RESUMO

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Transtornos Plaquetários/genética , Clonagem Molecular , Dislexia/genética , Eritrócitos Anormais , Células HEK293 , Humanos , Ictiose/genética , Espectroscopia de Ressonância Magnética , Transtornos de Enxaqueca/genética , Miose/genética , Modelos Moleculares , Fadiga Muscular/genética , Mutação/genética , Conformação de Ácido Nucleico , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Baço/anormalidades
2.
Cell Mol Life Sci ; 78(19-20): 6645-6667, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34498097

RESUMO

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Legend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Mutação Puntual/genética
3.
Protein Sci ; 32(3): e4571, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36691702

RESUMO

Stromal interaction molecule 1 (STIM1) resides in the endoplasmic reticulum (ER) membrane and senses luminal calcium (Ca2+ ) concentration. STIM1 activation involves a large-scale conformational transition that exposes a STIM1 domain termed "CAD/SOAR", - which is required for activation of the calcium channel Orai. Under resting cell conditions, STIM1 assumes a quiescent state where CAD/SOAR is suspended in an intramolecular clamp formed by the coiled-coil 1 domain (CC1) and CAD/SOAR. Here, we present a structural model of the cytosolic part of the STIM1 resting state using molecular docking simulations that take into account previously reported interaction sites between the CC1α1 and CAD/SOAR domains. We corroborate and refine previously reported interdomain coiled-coil contacts. Based on our model, we provide a detailed analysis of the CC1-CAD/SOAR binding interface using molecular dynamics simulations. We find a very similar binding interface for a proposed domain-swapped configuration of STIM1, where the CAD/SOAR domain of one monomer interacts with the CC1α1 domain of another monomer of STIM1. The rich structural and dynamical information obtained from our simulations reveals novel interaction sites such as M244, I409, or E370, which are crucial for STIM1 quiescent state stability. We tested our predictions by electrophysiological and Förster resonance energy transfer experiments on corresponding single-point mutants. These experiments provide compelling support for the structural model of the STIM1 quiescent state reported here. Based on transitions observed in enhanced-sampling simulations paired with an analysis of the quiescent STIM1 conformational dynamics, our work offers a first atomistic model for CC1α1-CAD/SOAR detachment.


Assuntos
Canais de Cálcio , Cálcio , Humanos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células HEK293 , Simulação de Acoplamento Molecular , Proteína ORAI1/metabolismo , Domínios Proteicos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo
4.
Sci Signal ; 16(771): eadd0509, 2023 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-36749824

RESUMO

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/metabolismo , Canais de Cálcio/metabolismo , Aminoácidos/metabolismo , Mutação , Retículo Endoplasmático/metabolismo , Molécula 1 de Interação Estromal/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Proteína ORAI1/metabolismo , Cálcio/metabolismo
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