Robert A. Saxton

The Sestrins Interact with GATOR2 to Negatively Regulate the Amino-Acid-Sensing Pathway Upstream of mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway.

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway.

From sensing leucine to metabolic control The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson et al. identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton et al. describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett et al. determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target. Science, this issue p. 43, p. 48, p. 53; see also p. 25 A crystal structure reveals how cells sense leucine for metabolic regulation. [Also see Perspective by Buel and Blenis] Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.

Mechanism of arginine sensing by CASTOR1 upstream of mTORC1.

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 acids. 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 GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). 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.

SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway

SAMTOR joins the family The amino acid methionine is widely appreciated to have interesting effects on animal physiology. Diets low in methionine increase longevity and overall health, particularly glucose homeostasis. Gu et al. describe a potential molecular link between the effects of methionine restriction and the growth controller mTOR complex 1 (mTORC1), a well-validated regulator of life span and health span in many organisms. They identify a protein that they named SAMTOR as a component of the nutrient-sensing pathway upstream of mTORC1. SAMTOR directly binds S-adenosylmethionine (SAM), a metabolite made from methionine, and is necessary for regulating mTORC1 in response to methionine. Science, this issue p. 813 SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling. mTOR complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple environmental cues. Nutrients signal via the Rag guanosine triphosphatases (GTPases) to promote the localization of mTORC1 to the lysosomal surface, its site of activation. We identified SAMTOR, a previously uncharacterized protein, which inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. We found that the methyl donor S-adenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 μM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling.

The apo-structure of the leucine sensor Sestrin2 is still elusive

The unliganded Sestrin2 structure awaits discovery. An elusive apo-structure for Sestrin2 The protein complex called mTORC1 integrates growth factor signals and nutrient status to control cell growth and metabolism. The molecular mechanism by which mTORC1 activity is controlled by nutrients has been an active and controversial area of research. Castor1 and Sestrin2 have been reported to function as an arginine sensor and a leucine sensor, respectively. Saxton et al. provide evidence that all of the available Sestrin2 crystals and crystals that they generated in the absence of exogenous leucine contain a bound ligand that is most likely leucine. These data indicate that the unbound apo-structure awaits determination. Not only will a true apo-structure be informative, but crystals containing Sestrin2 and other binding partners will as well. Thus, the mechanism of exactly how amino acids control mTORC1 activity awaits further study. Sestrin2 is a GATOR2-interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine [Protein Data Bank (PDB) ID, 5DJ4] and demonstrated that mutations in the leucine-binding pocket that alter the affinity of Sestrin2 for leucine result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID, 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. On the basis of this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and that Sestrin2 may not be a sensor for leucine. We show that simple analysis of the reported “apo”-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported apo-structure with leucine eliminated the large Fobs-Fcalc difference density at this position and improved the working and free R factors of the model. Consistent with this result, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contained electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive.