Shrestha Nisha, Hye-Ryong Shim Ann, Maneshi Mohammad Mehdi, See-Wai Yeung Priscilla, Yamashita Megumi, Prakriya Murali
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
J Biol Chem. 2022 Aug;298(8):102157. doi: 10.1016/j.jbc.2022.102157. Epub 2022 Jun 17.
Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca sensor and activator of Orai1 channels. In resting cells with replete Ca stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1-CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1-CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1-CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease-linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.
基质相互作用分子1（STIM1）是一种广泛表达的蛋白质，作为内质网（ER）钙传感器和Orai1通道的激活剂发挥作用。在钙储存充足的静息细胞中，由卷曲螺旋1（CC1）结构域与STIM1的CRAC激活结构域（CAD）相互作用形成的抑制钳有助于使STIM1保持静止状态。内质网钙储存耗尽后，制动解除，使CAD从内质网膜延伸开来，并使其能够激活Orai1通道。然而，对形成抑制钳的CC1-CAD相互作用的分子决定因素尚未完全了解。在这里，我们结合活细胞荧光共振能量转移（FRET）分析进行丙氨酸诱变，以研究CC1和CAD中调节抑制钳的残基。我们的结果表明，除了先前在CC1α1和CC3中确定的热点外，CC2中的几个疏水残基和CAD的顶端区域对于CC1-CAD相互作用至关重要。这些残基的突变会使CC1-CAD抑制钳松弛，从而在钙储存充足的细胞中将CAD从CC1中释放出来。相比之下，改变疏水残基L265和L273会加强钳制作用，以防止STIM1激活。包含STIM1的失活结构域有助于稳定几个突变体中的CC1-CAD相互作用，以防止STIM1自发激活。此外，CC3中与人类疾病相关的突变R426C会影响钳制作用，但也会损害Orai1的结合，从而抑制CRAC通道激活。这些结果确定了STIM1的CC2、顶端和失活结构域区域是STIM1激活的重要决定因素。
