New insights into GATOR2-dependent interactions and its conformational changes in amino acid sensing

Abstract Eukaryotic cells coordinate growth under different environmental conditions via mechanistic target of rapamycin complex 1 (mTORC1). In the amino-acid-sensing signalling pathway, the GATOR2 complex, containing five evolutionarily conserved subunits (WDR59, Mios, WDR24, Seh1L and Sec13), is required to regulate mTORC1 activity by interacting with upstream CASTOR1 (arginine sensor) and Sestrin2 (leucine sensor and downstream GATOR1 complex). GATOR2 complex utilizes β-propellers to engage with CASTOR1, Sestrin2 and GATOR1, removal of these β-propellers results in substantial loss of mTORC1 capacity. However, structural information regarding the interface between amino acid sensors and GATOR2 remains elusive. With the recent progress of the AI-based tool AlphaFold2 (AF2) for protein structure prediction, structural models were predicted for Sentrin2-WDR24-Seh1L and CASTOR1-Mios β-propeller. Furthermore, the effectiveness of relevant residues within the interface was examined using biochemical experiments combined with molecular dynamics (MD) simulations. Notably, fluorescence resonance energy transfer (FRET) analysis detected the structural transition of GATOR2 in response to amino acid signals, and the deletion of Mios β-propeller severely impeded that change at distinct arginine levels. These findings provide structural perspectives on the association between GATOR2 and amino acid sensors and can facilitate future research on structure determination and function.

Figure S1.An overview of the Sestrin2-WDR24-Seh1L complex models.(A) The superimposition of the five models.(B) A diagram of the predicted IDDT scores.Above the diagram, the corresponding WDR24, Sestrin2 and Seh1L positions are indicated.

Figure S2 .
Figure S2.Analysis of the interface between Sestrin2 and WDR24 Nβ. (A) Atom-atom interactions within 5 Å between WDR24 1-400 and Sestrin2, or Seh1L and Sestrin2.Any interactions closer than 5 Å are presented as black dots.Online COCOMAPS tool was used to generate the contacts.(B) Sequence alignments of WDR24 (residues 40 to 170) from cross-kingdom species.(C) Flex ddG computations of saturation mutation on Sestrin2 (L351, D346 and D364) and WDR24 (R46, K120 and R167).(D) Two alternative binding conformations of WDR24 K120 .Hydrogen bonds are indicated as yellow dotted lines.(E) Free energy landscape of

Figure S3 .
Figure S3.Sequence alignment of Sestrin2 with different homologous species.ESPript 3.0 was used to generate the alignment.The secondary structure elements of human Sestrin2 are placed on the top of the alignment where the NTD of Sestrin2 is coloured in green and the CTD is coloured in magenta.The residues selected in this

Figure S5 .
Figure S5.Assessment of the predicted model of Mios Nβ -CASTOR1.(A) An overview of the Predicted Aligned Error plot from the five models of Mios Nβ -CASTOR1 complex.The corresponding chains are indicated.Within the plot, the black lines delineate the area of individual protein of the complex.(B) Cartoon representation of the top-ranked model colour-coded by the pLDDT values.(C) Sequence alignment for CASTOR1 (residues 140 to 200).(D) Sequence alignment for Mios.The conserved residues marked blue triangles in (C, D) were selected for co-IP experiments.(E) Flex ddG computations of saturation mutations on Mios (K64, R137 and R206) and CASTOR1 (E146, D190 and E192).

Figure S6 .
Figure S6.The 1st vibrational mode of GATOR2 NMA.Displacement of each Cα atom in the 7th normal mode, and the corresponding subunits are indicated above.Two β-sheets of WDR24 were shown to contribute the highest level of displacements as shown in Movie S1 and S2.

Figure S7 .
Figure S7.Effects of leucine and Sestrin2 on the conformational changes of GATOR2.(A) The combined expression of CFP or YFP showing unaffected complex formation of GATOR2 with METAP2 used as a negative control.The results are representative of three independent experiments.(B) Representative pictures of HEK-293T cells expressing YFP-or CFP-conjugated proteins.(C) Assessment of FRET efficiency of GATOR2 in response to leucine.Error bars mean ± sd, n≥15.* = p<0.05,** = p<0.01,*** = p<0.001,**** = p<0.0001.

Figure S8 .
Figure S8.Effects of Mios-Nβ deletion on the conformational change in GATOR2.(A) Mios-Nβ deletion showing no effect on the GATOR2 integrity; WDR24-Nβ deletion partially impairs the formation of GATOR2 complex.The results are representative of three independent experiments.(B) Aligning AF2 model of CASTOR1-Mios Nβ with the architecture of GATOR2.Panorama view of CASTOR1-GATOR2 complex was visualized via USCF ChimeraX program.(C) A graphic model for the conformational change of GATOR2 in response to amino acids.