1. Evaluation Summary:

    This study will be of interest to structural biologists, enzymologists, and cell biologists. The kinase complex mTORC1, a master regulator of cell growth, can be inhibited by another protein, DEPTOR. This protein is of general interest for several reasons, including the hope that understanding how DEPTOR works will lead to new strategies for therapeutically tuning mTORC1 activity. This study provides insights into the binding and inhibitory effects of DEPTOR on mTORC1. Solving a few technical questions will improve the work.

    (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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  2. Reviewer #1 (Public Review):

    mTORC1 activity promotes anabolic growth and suppresses autophagy. Because mTORC1 integrates growth signals, nutrient concentrations, and other variables to coordinate metabolism with growth and division, mTORC1 dysfunction contributes to cancers, metabolic derangements, autoimmune and neurological disorders. DEPTOR is an endogenous protein inhibitor of mTORC1 that is of general interest for several reasons, including the hope that understanding how DEPTOR works will lead to new strategies for therapeutically tuning mTORC1 activity.

    In this study, Heimhalt et al. succeeded in providing new structure/function insights into the binding and inhibitory effects of DEPTOR on mTORC1. Using in vitro kinase assays with all purified components, electron cryo-microscopy, NMR, and homology models the authors report that DEPTOR binds and partially inhibits mTORC1 via two distinct surfaces. Remarkably, DEPTOR can only inhibitor mTORC1 activity by <50%, and its inhibitory activity appears to depend at least in part on a slow, allosteric conformational change and to be limited by a negative feedback loop. Specifically, the authors build on prior work to show that DEPTOR is a phosphorylation substrate of mTORC1 and that phosphorylated DEPTOR cannot inhibit mTORC1. The authors speculate that the partial and self-limiting inhibition of mTORC1 by DEPTOR evolved so that DEPTOR can "blunt" mTORC1 activity without increasing tumorigenic PI3K signaling due to loss of mTORC1 feedback inhibition.

    A central message of the manuscript is that, in contrast to previous cell-based studies, the authors find that DEPTOR requires both its PDZ domain and adjacent "long linker" for inhibition. The authors propose that the linker's interaction with the FRB domain of mTORC1 is crucial to the partial inhibition mechanism. As with other studies of mTORC1 complexes by cryo-EM, the maps included in this study are challenging to interpret, especially around the low-resolution periphery where DEPTOR's domains may bind. Hence, the authors used a battery of additional techniques, including HDX-MS, NMR, and homology modeling, to bolster their interpretations. However, the binding mode and role of DEPTOR's linker region remain underdetermined and are the focus of detailed recommendations to the authors. Pending the resolution of the technical questions, this study should make an impactful contribution of interest to structural biologists, kinase enzymologists, and cell biologists.

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  3. Reviewer #2 (Public Review):

    The manuscript by Heimhalt et al uses biochemical reconstitution, structural and biophysical techniques to shed light on the mTORC1 subunit, DEPTOR, and its regulatory roles toward mTORC1-dependent signaling. The authors report that DEPTOR associates with the mTOR protein via two domains, the PDZ and an unstructured linker, binding to the FAT and FRB domains, respectively. This bipartite interaction appears critical for maintaining DEPTOR bound and for partially inhibiting substrate engagement, likely via an allosteric mechanism (as opposed to direct substrate competition). Interestingly, DEPTOR-mediated inhibition is stronger on active mTORC1 than on the inactive (non-RHEB bound) complex, a claim supported by both biochemical and structural considerations. Finally, as part of a regulatory feedback, DEPTOR phosphorylation by mTORC1 in the linker region decreases DEPTOR ability to bind to and inhibit mTORC1. Overall, this is an interesting and well executed manuscript that sheds light on an important component of the mTORC1 complex. The experiments are of high quality and support the main claims.

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  4. Reviewer #3 (Public Review):

    It has been known for more than 10 years that DEPTOR is a negative regulator of mTORC1 and it has more recently come to light that this regulation may be particularly important in disease states such as multiple myeloma. In spite of the great interest in this topic, it has remained unclear exactly how DEPTOR interacts with and regulates mTORC1. It is therefore noteworthy that this study make significant progress towards defining the basis for DEPTOR-dependent inhibition of mTORC1 through a compelling combination of structural and biochemical approaches. The results define a novel bipartite binding mechanism for DEPTOR interactions with mTOR and characterize the basis for partial inhibition of mTOR by DEPTOR. Of further interest is the elucidation of a feedback loop whereby mTORC1 phosphorylates DEPTOR which suppresses the ability of DEPTOR to inhibit mTORC1. The overall quality of the data is high and the authors have offered a balanced and thoughtful description of their results and of how these findings can be integrated into existing knowledge in this field. This is a very rare example of a manuscript that where I cannot identify any major weakness.

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