Molecular Mechanisms of Orai1 Channel Activation by STIM1

Yeung, Priscilla See-Wai

doi:https://doi.org/10.21985/n2-6g4z-0w97

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Molecular Mechanisms of Orai1 Channel Activation by STIM1

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Store-operated Ca2+ entry through Orai1 channels mediate transcriptional, proliferative, and effector cell programs in many cells and are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor, STIM1, to cytoplasmic sites on Orai1. Mutations in Orai1 that block channel activation or evoke constitutive channel activity are known to cause debilitating diseases in humans such as immunodeficiency, autoimmunity, myopathy, and thrombocytopenia. However, our understanding of the underlying molecular mechanisms of these diseases is limited by gaps in our fundamental knowledge of how Orai1 channels are gated. In particular, although atomic level details of Orai structure including the pore and putative ligand binding domains are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. This dissertation synthesizes our work over the past four years on the molecular mechanisms of Orai1 gating by STIM1. Chapter 2 summarizes a project (led by Megumi Yamashita) in which we identified residue F99 as a key component of the channel gate that is activated through pore helix rotation following STIM1 binding. In Chapter 3, I present my work on the roles of the transmembrane domains (TMs) in relaying the gating signal from STIM1 binding to the pore. We used scanning mutagenesis to identify fifteen residues in TMs 1-4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues, and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels. Chapter 4 discusses the potential role of human mutation Orai1 L138F in channel activation and inactivation. In Chapter 5, I present preliminary results and hypotheses about specialized features in the Orai1 protein that may facilitate pore opening, including the presence of water crevices behind the pore helices and the M101 latch that stabilizes F99 into the rotated state.

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