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S-adenosylmethionine sensor upstream of TORC1: Biological Overview | References

The mechanistic target of rapamycin-mLST8-raptor complex (mTORC1) functions as a central regulator of cell growth and metabolism in response to changes in nutrient signals such as amino acids. SAMTOR is an S-adenosylmethionine (SAM) sensor, which regulates the mTORC1 activity through its interaction with the GTPase-activating protein activity toward Rags-1 (GATOR1)-KPTN, ITFG2, C12orf66 and SZT2-containing regulator (KICSTOR) complex. This paper reports the crystal structures of Drosophila melanogaster SAMTOR in apo form and in complex with SAM. SAMTOR comprises an N-terminal helical domain and a C-terminal SAM-dependent methyltransferase (MTase) domain. The MTase domain contains the SAM-binding site and the potential GATOR1-KICSTOR-binding site. The helical domain functions as a molecular switch, which undergoes conformational change upon SAM binding and thereby modulates the interaction of SAMTOR with GATOR1-KICSTOR. The functional roles of the key residues and the helical domain are validated by functional assays. These structural and functional data together reveal the molecular mechanism of the SAM sensing of SAMTOR and its functional role in mTORC1 signaling (Tang, 2022).

The mechanistic target of rapamycin (mTOR) is a multifunctional kinase that plays important roles in embryonic development, aging, tumorigenesis, diabetes, and neurodegenerative diseases. In mammalian cells, mTOR forms two functionally distinct complexes, the mTOR-mLST8-Raptor complex (mTORC1) and the mTOR-mLST8-Rictor complex (mTORC2). In response to environmental conditions of energy, nutrients, and extracellular growth factors, mTORC1 modulates the anabolic pathway and promotes the initiation and elongation of protein translation through directly phosphorylating specific substrates such as S6 kinase 1 (S6K1) and 4E binding protein 1 (4EBP1). In addition, mTORC1 suppresses the catabolic pathway through inhibition of autophagy and lysosome biogenesis (Tang, 2022).

Two complementary parallel signaling pathways work together to render full activation of mTORC1 at the lysosomal membrane. On one hand, amino acids induce the conversion of small guanosine triphosphatases (GTPases) Ras-related GTP-binding protein A (RagA) to RagD to the active nucleotide-bound state, i.e., the guanosine triphosphate (GTP)-loaded state of RagA/B and the guanosine diphosphate-loaded state of RagC/D. After that, a lysosomal multisubunit machinery comprising the vacuolar-type adenosine triphosphatase (v-ATPase), the pentameric Ragulator complex, and the active Rag GTPases recruits mTORC1 to the lysosomal surface. On the other hand, the growth factor-stimulated kinase Akt phosphorylates and then inhibits the tuberous sclerosis complex, which acts as a GTPase-activating protein (GAP) for the small GTPase Rheb at the lysosomal membrane, where the active GTP-bound Rheb can fully activate mTORC1 (Tang, 2022).

Rag GTPases function as obligate heterodimers such that RagA/B interacts with RagC/D through their C-terminal roadblock domains, and their N-terminal GTPase domains dictate their interactions with the mTORC1 unique component Raptor. The GATOR1 complex, comprising three subunits [Nprl2, Nprl3, and DEP domain-containing protein 5 (DEPDC5)], functions upstream of Rag GTPases as a GAP for RagA/B to inactivate mTORC1 when amino acids are deficient. The KICSTOR scaffolding complex, consisting of four subunits [Kaptin, integrin-alpha FG-GAP repeat-containing protein 2 (ITFG2), C12orf66, and SZT2], tethers GATOR1 to the lysosomal surface and facilitates the interaction between GATOR1 and Rag GTPases. The GATOR2 complex, consisting of five subunits [WD repeat domain 59 (WDR59), WD repeat domain 24 (WDR24), meiosis regulator for oocyte development (MIOS), SEH1 like nucleoporin (SEH1L), and SEC13], functions upstream of GATOR1 as an inhibitor of GATOR1 and thus a positive regulator of mTORC1. The cytoplasmic leucine and arginine activate mTORC1 through regulating the dynamic interplay of GATOR1 and GATOR2. Upon leucine/arginine deprivation, the cytoplasmic leucine sensors Sestrin1/2 and SAR1B or the arginine sensor CASTOR1 (cytosolic arginine sensor for mTORC1 subunit 1) interact with GATOR2 and block the GATOR1-GATOR2 interaction, releasing the GAP activity of GATOR1 for RagA/B, and the binding of leucine to Sestrin1/2 and SAR1B or arginine to CASTOR1 impairs the sensor's interaction with GATOR2, leading to inactivation of GATOR1 and thus activation of mTORC1 (Tang, 2022).

Similar to leucine and arginine, methionine regulates mTORC1 in a Rag GTPase-dependent manner. However, the direct cytoplasmic methionine sensor in mTORC1 signaling has not been found so far. Recently, an S-adenosylmethionine (SAM)-binding protein SAMTOR (or C7orf60) was identified as a negative regulator of mTORC1, which functions upstream of Rag GTPases, GATOR1, and KICSTOR: SAMTOR can interact with GATOR1 to prompt the function of GATOR1 and/or KICSTOR in the absence of SAM, and suppresses Rag GTPases and mTORC1; with the supply of SAM, the binding of SAM to SAMTOR disrupts the interaction of SAMTOR with GATOR1-KICSTOR, leading to the inhibition of the GATOR1 GAP activity and, thus, the activation of mTORC1. SAM is synthesized from methionine and adenosine triphosphate (ATP), and the cellular SAM level is directly correlated with methionine. The silencing of methionine adenosyltransferase MAT2A, which catalyzes the synthesis of SAM from methionine and ATP, decreases the expression of SAMTOR and the activation of mTORC1. Loss of SAMTOR prevents the inhibition of mTORC1 caused by methionine starvation. Thus, SAMTOR serves as a cytoplasmic SAM sensor in the SAM/methionine-mediated mTORC1 signaling (Tang, 2022).

To illuminate the molecular mechanism of the functional role of SAMTOR in the SAM/methionine-mediated mTORC1 signaling, this study determined the crystal structures of Drosophila melanogaster SAMTOR (dSAMTOR) in apo and SAM- and S-adenosyl-l-homocysteine (SAH)-bound forms. Structural analysis shows that SAMTOR comprises an N-terminal helical domain and a C-terminal class I SAM-dependent methyltransferase (MTase) domain. The ligand (SAM/SAH) binds to the MTase domain and makes extensive hydrogen-bonding and hydrophobic interactions with the surrounding residues. The functional roles of the key residues involved in ligand binding are validated by mutagenesis and biochemical assays. In addition, it was found that the N-terminal helical domain exhibits a high flexibility and acts as a molecular switch in response to SAM/SAH binding. In the absence of SAM/SAH, the helical domain is positioned away from the ligand-binding site, allowing SAMTOR to interact with GATOR1-KICSTOR. The binding of SAM/SAH appears to induce conformational change of the helical domain to cover the ligand-binding site, blocking the interaction of SAMTOR with GATOR1-KICSTOR. The structural and functional data together provide insight into the molecular mechanism of SAM/SAH sensing by SAMTOR in the SAM/methionine-mediated mTORC1 signaling (Tang, 2022).

The sulfur-containing amino acid methionine presents a key metabolite in many aspects of mammalian physiology, including translation, epigenetics, cell proliferation, and various signaling pathways. In metazoans, the mTORC1 signaling pathway senses the cellular level of the methionine metabolite SAM rather than methionine through the SAM sensor SAMTOR to modulate the anabolic and catabolic metabolisms. This work determined the crystal structures of the MTase domain of dSAMTOR in SAM- and SAH-bound forms and the full-length V66W/E67P mutant dSAMTOR in apo form. Structural analyses reveal that dSAMTOR comprises an N-terminal helical domain and a C-terminal MTase domain. The helical domain consists of three α helices (αA to αC) and exhibits a high flexibility. The MTase domain adopts a class I MTase fold supplemented with some auxiliary structure elements. In the SAM- and SAH-bound dSAMTOR MTase structures, the SAM and SAH bind to the MTase domain in a similar binding manner and with comparable binding affinities. The MTase domain of dSAMTOR and the full-length dSAMTOR exhibit comparable binding affinities with the ligands, suggesting that the helical domain is not directly involved in ligand binding. Comparison of the SAM-bound MTase domain and the apo V66W/E67P mutant structures shows that ligand binding does not induce notable conformational changes in the overall structure of the MTase domain except a few minor conformational changes at the ligand-binding site. The ligand makes extensive hydrophilic and hydrophobic interactions with several conserved residues, and mutations of these residues impair ligand binding in vitro. Selective mutations with deficient ligand binding also abolish the SAM-sensing ability of hSAMTOR and the mutants could bind to GATOR1-KICSTOR both in the absence and in the presence of SAM in vivo. These results reveal the molecular mechanism of SAM sensing and binding by SAMTOR (Tang, 2022).

Recently, several direct amino acid sensors upstream of mTORC1 including the arginine sensor CASTOR1 and the leucine sensor Sestrins2 have been identified and the crystal structures of these sensors have been solved. The structural and functional data together have revealed the molecular mechanisms about how CASTOR1 and Sestrins2 sense and bind the ligands. However, as ligand binding does not induce substantial conformational changes of the proteins, the molecular mechanisms about how these sensors function as molecular switches upon ligand binding to interact with the downstream partners and then further regulate mTORC1 signaling remain unclear. On the basis of the structural and functional data of SAMTOR together with the structural comparison with the AlphaFold predicted SAMTOR structure and the structures of several MTases, a working model is proposed for how SAMTOR functions as a molecular switch upon SAM binding to regulate mTORC1 signaling as follows. The C-terminal MTase domain of SAMTOR contains both the ligand-binding site and the GATOR1-KICSTOR-binding site. The MTase domain of SAMTOR alone has the ligand binding ability as well as the GATOR1-KICSTOR binding ability in both the presence and absence of the ligand. In other words, it has the ability to sense the SAM level but lacks the switch function to regulate mTORC1 signaling upon SAM binding. On the other hand, while the N-terminal helical domain of SAMTOR is not required for the interactions with the ligand and GATOR1-KICSTOR, it has a high flexibility and functions as a molecular switch to turn on and off the binding ability of the MTase domain with GATOR1-KICSTOR and subsequently mTORC1 signaling. In the absence of SAM, the helical domain is positioned distantly from the ligand-binding site with a flexible conformation, and hence the potential binding site for GATOR1-KICSTOR is exposed and the MTase domain can interact with GATOR1-KICSTOR, leading to the inhibition of mTORC1 signaling. When SAM binds to SAMTOR, the helical domain is positioned on the top of the ligand-binding site and the conformation is stabilized by SAM binding, and hence the potential binding site for GATOR1-KICSTOR is blocked and thus the MTase domain cannot interact with GATOR1-KICSTOR, leading to the activation of mTORC1 signaling (Tang, 2022).

Biochemical and structural data show that dSAMTOR can bind SAH and SAM with comparable binding affinities, and SAH binds to the MTase domain with almost identical interactions with the surrounding residues as SAM. The previous cell biology data also show that like SAM, the binding of SAH to hSAMTOR can disrupt the interaction of hSAMTOR with GATOR1-KICSTOR and inhibit the GATOR1 GAP activity in mTORC1 signaling. As the concentrations of SAM and SAH in cells are estimated to be at similar levels, these results suggest that SAH might be able to regulate the function of SAMTOR in mTORC1 signaling in the same manner as SAM (Tang, 2022).

Previous studies show that the arginine sensor CASTOR1 consists of four tandem ACT domains and structurally resembles the C-terminal allosteric domains of aspartate kinases, suggesting that CASTOR1 may have evolved from the ancient aspartate kinase but lost the N-terminal kinase domain during evolution. The leucine sensor Sentrin2 is a homolog of bacterial reductase Ahpd but lacks a conserved cysteine required for the catalytic activity. So far, whether Senstrin2 has a reductase activity remains elusive. SAMTOR contains a conserved class I MTase domain, which is usually known to bind and exert the catalytic activity on DNA or RNA during the regulation of gene expression, repair of mutations, and protection against bacterial restriction enzymes. Sequence alignment shows that the SAMTOR proteins have a moderate sequence similarity (~11% identity) to Saccharomyces cerevisiae Bmt2, an MTase responsible for the m1A modification of 25S ribosomal RNA (rRNA). Structural comparison of dSAMTOR with the small RNA MTase Hen1 (PDB code 3HTX) and the long noncoding RNA MTase MePCE (methyl phosphate capping enzyme) (PDB code 6DCB) of class I MTases shows that both Hen1 and MePCE have a positively charged surface cleft around the active site of the MTase domain to bind the RNA substrate. In contrast, the MTase domain of dSAMTOR contains no such kind of positively charged surface cleft; instead, the electrostatic surface around the ligand-binding site is largely negatively charged or hydrophobic, which is apparently unfavorable for the binding of an RNA or DNA substrate, suggesting that SAMTOR is unlikely an RNA or DNA MTase. Nevertheless, whether SAMTOR has an MTase activity on proteins remains elusive and warrants further study (Tang, 2022).

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Tang, X., Zhang, Y., Wang, G., Zhang, C., Wang, F., Shi, J., Zhang, T. and Ding, J. (2022). Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling. Sci Adv 8(26): eabn3868. PubMed ID: 35776786

date revised: 6 April 2023

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