Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

Abstract

Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.

STIM Proteins: An Ever-Expanding Family

“…One contact is formed between L258 and L261 of α1 to I290 and A293 of α2, while another one was found between L248 and L251 of α1 and L300 and L303 of α2 (Figure 2a, right panel) [22]. Interestingly and as already mentioned above, L248, L251, L258, and L261 all disrupt the CC1α1-CC3 clamp upon mutation [16,[18][19][20]68]. Since this match clearly hints towards an effect of the CC1α1-CC1α2 interactions on the coiled coil clamp, Rathner et al disrupted the interactions by targeted double point mutations of the hydrophobic CC1α2 residues to hydrophilic serines.…”

“…However, whether these actually are the directly interacting amino acids has not yet been clarified. The hypothesized CC3 counterparts are L416, V419, and L423 [16,[18][19][20]68]. Judging from the evidence, these leucines and valines could form the clamp through intramolecular hydrophobic interactions, which are sufficiently strong to stabilize the STIM1 inactive state while still allowing a conformational switch to occur.…”

The Orai Pore Opening Mechanism

“…Structural studies [ 34 , 35 , 36 ] complemented by a series of molecular dynamics (MD) simulations [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ] revealed new detailed insights into the understanding of the STIM1 and Orai1 activation mechanism. So far, for STIM1, only the structures of N- and C-terminal STIM1 fragments are available [ 46 , 47 , 48 , 49 , 50 ].…”

Engineering supramolecular organizing centers for optogenetic control of innate immune responses

“…Ca2+ influx and NFAT signaling in cells of the immune system and tissue morphogenesis (21,(26)(27)(28)(29)(30). Inspired by these engineering strategies, we have developed a light-stimulable Photo-SMOC (MyDDosome and MAVSome) system and a light-controllable IRF3 activation device to photo-tune both the upstream and (which was not certified by peer review) is the author/funder.…”