Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1
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Evaluation Summary:
This study uses complementary approaches to advance our mechanistic understanding of STIM1 activation, with elegant single molecule methods providing new details on STIM1 structure and dynamics. Full length STIM1 in a cellular environment was probed by crosslinking, but the same has not yet been possible with single-molecule Förster resonance energy transfer (smFRET).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
The dimeric ER Ca 2+ sensor STIM1 controls store-operated Ca 2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca 2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it toward the plasma membrane to activate Orai and SOCE after store depletion.
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Reviewer #3 (Public Review):
In this study, van Dorp et al. provide new insights into the structure of the C-terminus of STIM1 in the quiescent as well as the active state. By using extensive smFRET and protein crosslinking techniques, the authors substantially advanced our understanding of STIM1 cytosolic domains orientation and revealed inter- and intramolecular interactions within a STIM1 dimer. Structures have been derived for both STIM1 resting and activated state. Altogether, this study substantially contributes to a mechanistic and structural understanding of the STIM1 activation process, and it paths the way for the comprehensive dynamic resolution of conformational transitions from the inactive to the fully active state.
The single molecule studies represent a very elegant approach to derive novel details on STIM1 structure and …
Reviewer #3 (Public Review):
In this study, van Dorp et al. provide new insights into the structure of the C-terminus of STIM1 in the quiescent as well as the active state. By using extensive smFRET and protein crosslinking techniques, the authors substantially advanced our understanding of STIM1 cytosolic domains orientation and revealed inter- and intramolecular interactions within a STIM1 dimer. Structures have been derived for both STIM1 resting and activated state. Altogether, this study substantially contributes to a mechanistic and structural understanding of the STIM1 activation process, and it paths the way for the comprehensive dynamic resolution of conformational transitions from the inactive to the fully active state.
The single molecule studies represent a very elegant approach to derive novel details on STIM1 structure and dynamics. Utilization of these developed smFRET protein probes of ctSTIM1 in the interaction with Orai1, either reconstituted or even in living cells, would be phantastic, but certainly experimentally challenging based on the low fluorescent background required to resolve single molecule FRET.
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Reviewer #2 (Public Review):
Although the major activation steps and general mechanistic underpinnings of SOCE have been reported in a flurry of literatures, they are largely descriptive and lack quantitative information. One topic of greatest interest to the CRAC channel field is the structural basis of CC1-CAD/SOAR-mediated STIM1 autoinhibition. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, Dorp et al provides a binding model for the CC1-CAD interaction. This model explains the role of CC1 in STIM1 activation, and delineates the activation process of STIM1 CT. It also clarifies the controversy on the two varying structures regarding the packing of the CAD/SOAR domain by favoring the X-ray structure over the NMR structure. The conclusions of this paper are mostly well supported by …
Reviewer #2 (Public Review):
Although the major activation steps and general mechanistic underpinnings of SOCE have been reported in a flurry of literatures, they are largely descriptive and lack quantitative information. One topic of greatest interest to the CRAC channel field is the structural basis of CC1-CAD/SOAR-mediated STIM1 autoinhibition. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, Dorp et al provides a binding model for the CC1-CAD interaction. This model explains the role of CC1 in STIM1 activation, and delineates the activation process of STIM1 CT. It also clarifies the controversy on the two varying structures regarding the packing of the CAD/SOAR domain by favoring the X-ray structure over the NMR structure. The conclusions of this paper are mostly well supported by data. The only minor concern is to reconcile some of the conflicting results (regarding the relative positions of some residues used in the crosslinking study, as well as the CC1-alpha 1 helix), made between this study and a recent structural study, i.e., the NMR solution structure of CC1 reported by the Romanin/Muller's groups (PMID: 33106661). Overall, this study covers a timely topic to address a long-standing question in the ORAI-STIM signaling field, i.e., the structural basis of CC1-CAD association that keeps STIM1 largely quiescent in the resting condition. This work, regarded by this reviewer as a "tour-de-force" by meticulously scanning through many key residues within the multiple CC1/CAD helices, certainly warrants immediate publication.
Notable strengths:
smFRET is increasingly being used to determine distances, structures, and dynamics of biomolecules. Full length STIM1 and STIM1 C-terminus have been always difficult to obtain crystal structure due to its tendency for aggregation and the existence of large disordered regions. Herein, the authors selected smFRET as the major tool to overcome this hurdle and illuminated the CC1-CAD binding models to provide novel mechanistic insights into STIM1 auto-inhibition mediated by the intramolecular cis CC1-CAD association.
The efforts to extend crosslinking of ctSTIM1 to flSTIM1 are particularly commendable, moving one more step closer to the physiological scenario.
Minor weaknesses:
The authors proposed a CC1 model displaying "tandem connection of "CC1α1- CC1α2", that shows notable discrepancies with the recent CC1 NMR solution structure (PMID: 33106661). In the latter structure, the three helices are intertwined to form a bundle like structure. An in-depth discussion is certainly needed to clarify the difference. Some possibilities include: (i) Is this due to the artifact of the CC1 NMR structure (done in the presence of helix-stabilizing reagents)? (ii) is this due to the introduction of cysteine residues for the assays? (iii) is this due to absence of the CAD/SOAR part, or other regulatory components, in the solution structure? Repeating one or two key smFRET/crosslinking experiments in the presence of the similar buffer condition as in the NMR study would provide clues to these possibilities.
Another concern, very minor though, is regarding cysteine crosslinking flSTIM1 by 0.2 mM diamide. Will the addition of diamide cause undesired activation of STIM1 in the absence of cyclopiazonic acid?
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Reviewer #1 (Public Review):
The authors use smFRET and cross linking to constrain relative orientations of CC1-CC3 helices in STIM1 resting and active conformations. The data are excellent and especially because structures of full length STIM1 are currently lacking they paint an important picture of the structural basis for STIM1 activation. The number of smFRET pairs examined in the inactive state is fairly large and paints a good picture of the relative orientations of helices. In contrast, only a few pairs of sites were examined in activated STIM1 which paint a clear picture of CC1a1 dissociation from CC3, but the remaining postulated conformational changes during activation are inferred primarily from cross linking, and it would have been nice to probe those with smFRET as well. Nonetheless, the data yet provide very useful …
Reviewer #1 (Public Review):
The authors use smFRET and cross linking to constrain relative orientations of CC1-CC3 helices in STIM1 resting and active conformations. The data are excellent and especially because structures of full length STIM1 are currently lacking they paint an important picture of the structural basis for STIM1 activation. The number of smFRET pairs examined in the inactive state is fairly large and paints a good picture of the relative orientations of helices. In contrast, only a few pairs of sites were examined in activated STIM1 which paint a clear picture of CC1a1 dissociation from CC3, but the remaining postulated conformational changes during activation are inferred primarily from cross linking, and it would have been nice to probe those with smFRET as well. Nonetheless, the data yet provide very useful constraints on STIM1 conformational rearrangements that will be of great value to further structure-function studies.
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Evaluation Summary:
This study uses complementary approaches to advance our mechanistic understanding of STIM1 activation, with elegant single molecule methods providing new details on STIM1 structure and dynamics. Full length STIM1 in a cellular environment was probed by crosslinking, but the same has not yet been possible with single-molecule Förster resonance energy transfer (smFRET).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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