The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids1,2,3. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7).Arginine activates mTORC1 upstream of the Rag family of GTPases8, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1)9,10,11,12. However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor.
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We thank all members of the Sabatini and Schwartz laboratories for helpful insights. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work has been supported by grants from NIH (R01CA103866 and AI47389) and the US Department of Defense (W81XWH-07- 0448) to D.M.S. Fellowship support was provided by NIH to L.C. (F31 CA180271). D.M.S. is an investigator of the Howard Hughes Medical Institute.
D.M.S. is a founder and member of the Scientific Advisory Board, a paid consultant, and a shareholder of Navitor Pharmaceuticals, which is targeting for therapeutic benefit the amino-acid-sensing pathway upstream of mTORC1.
Reviewer Information Nature thanks L. Tong and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Expanded Multiple Sequence Alignment of CASTOR1 homologues from various organisms. Positions are coloured white to blue according to increasing sequence identity. Secondary structure features are labelled and coloured by ACT domain as in Fig. 1a.
Extended Data Figure 2 Dimerization-deficient CASTOR1 mutants bind arginine but fail to inhibit mTORC1 in cells.
a, The dimerization-deficient CASTOR1 Y207S and I202E mutants bind arginine in vitro. FLAG-immunoprecipitates prepared from HEK-293T cells transiently expressing indicated FLAG-tagged proteins were used in binding assays with [3H]Arginine as described in the Methods. Unlabelled arginine was included as a competitor where indicated. Values are mean ± s.d. for three technical replicates from one representative experiment. b, Dimerization-deficient CASTOR1 Y207S and I202E mutants fail to inhibit mTORC1. HEK-293T cells transiently expressing FLAG–S6K1 and HA-tagged wild-type, Y207S, or I202E CASTOR1 were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. FLAG- immunoprecipitates were prepared from lysates and analysed as in Fig. 1c. Phospho-S6K1 was used as an indicator of mTORC1 activity.
a, Comparison of the arginine-bound pocket of human CASTOR1 with a model of the pocket with lysine in place of arginine. Arginine and lysine stick representations are shown in yellow and orange, respectively. The distances in the lysine-bound model, 3.8 Å and 5.0 Å, are beyond the range of standard hydrogen bonds and salt bridges, respectively. ACT domains are labelled as in Fig. 1a. b, Chemical structures of arginine analogues used in Fig. 2e. Differences relative to l-arginine are highlighted in oranges boxes.
a, Multiple sequence alignment of human CASTOR1 and CASTOR2, highlighting differences in amino acid sequence that are in close proximity to arginine-binding residues in CASTOR1. b, The CASTOR1 HHV108–110QNI mutant constitutively binds GATOR2 in cells. HEK-293T cells transiently expressing HA–metap2 or the indicated HA-tagged CASTOR1 constructs were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. HA-immunoprecipitates were prepared and analysed as in Fig. 1c. c, The CASTOR1 HHV108–110QNI mutant displays reduced arginine-binding capacity in vitro. Binding assays were performed with the indicated CASTOR1 or CASTOR2 constructs and immunoprecipitates analysed as in Fig. 2c. Values are mean ± s.d. for three technical replicates from one representative experiment. d, Comparison of the CASTOR1 HHV108–110QNI mutant and wild-type CASTOR2. HEK-293T cells transiently expressing HA–metap2 or the indicated HA-tagged CASTOR1 or CASTOR2 constructs were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. HA-immunoprecipitates were prepared and analysed as in Fig. 1c.
Extended Data Figure 5 GATOR2-binding-deficient CASTOR1 mutants still bind arginine and homodimerize.
a, The CASTOR1 YQ118–119AA, D121A, E261A and D292A mutants bind arginine in vitro. FLAG-immunoprecipitates prepared from HEK-293T cells transiently expressing indicated FLAG-tagged proteins were used in binding assays with [3H]arginine as described in the Methods. Unlabelled arginine was included as a competitor where indicated. Values are mean ± s.d. for three technical replicates from one representative experiment. b, The CASTOR1 YQ118–119AA, D121A, E261A and D292A mutants dimerize in cells. HA-immunoprecipitates prepared from HEK293T-cells transiently expressing CASTOR1–FLAG and HA–metap2 or the indicated HA-tagged CASTOR1 constructs were analysed as in Fig. 1c.
a, Ribbon diagram views of human CASTOR1, AKeco (PDB ID: 2J0x) and AKsyn (PDB ID: 3L76), highlighting the different modes of dimerization. Aspartate kinases can dimerize through an interlocked-ACT domain conformation (as in AKeco) or through their kinase domains (AKsyn), both of which are distinct from the side-by-side ACT-domain dimerization in CASTOR1. b, View of AKeco depicting positions of residues R305, E346, and V347, which correspond to the positions of the GATOR2-interacting residues of CASTOR1.
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Saxton, R., Chantranupong, L., Knockenhauer, K. et al. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 536, 229–233 (2016). https://doi.org/10.1038/nature19079
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