Fig. 2: Cholesterol binding sites.
From: Structural insights into cholesterol sensing by the LYCHOS-mTORC1 pathway
a, b Electrostatic potential surfaces (a) and cartoon representation (b) of protomer A of the LYCHOSWT_C structure. CHS molecules are shown as sticks. c Details of the cholesterol binding site CBS1 of LYCHOSWT_C. CHS molecules are shown in green and sticks. Hydrophobic residues interacting with CBS1 are shown in sticks. Scaffold domain (PLD), transporter domain (PLD) and GLD are shown as gold, purple and deep sky blue, respectively. d Details of the cholesterol binding site CBS2. CHS2 and hydrophobic residues interacting with CBS2 are shown in sticks. Cartoon model for TM16 and TM11 are omitted for convenience to read. L660 from TM16 is shown in stick. e The molecular interaction between cholesterol molecules and LYCHOS. The interactions were revealed by 900 ns molecular dynamics simulation. Cholesterol (CLR) molecules are shown in green and sticks. Hydrophobic residues interacting with CBS1 are shown in sticks. Scaffold domain, transporter domain and GLD are shown as gold, purple and deep sky blue, respectively. Gly702 is shown in red. f The root mean square deviation (RMSD) of the cholesterol molecules in two CBSs during the 900 ns simulation for two trajectories. g Schematic of LYCHOS and GATOR1 modification design, fusing Rluc to the LYCHOS-C-terminus and linking YFP to the NPRL2-C-terminus of the GATOR1 heterotrimer via linker. In response to cholesterol stimulation, LYCHOS recruits GATOR1, and Rluc is brought into proximity with YFP, and energy resonance transfer occurs. h Detailed description of the Rluc and YFP fusion sites. Rluc is indicated in blue, and YFP is indicated in yellow. i–k Cholesterol dose-dependent profile of LYCHOS recruitment to GATOR1 after CBS mutations as determined by BRET. The values are reported as the mean ± SEM of three biologically independent experiments (n = 3). l Effect of CBS mutations on cholesterol-activated LYCHOS recruitment of GATOR1. Data from three biologically independent experiments (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05; ND, no detectable signal; ns no significant difference. Values are shown as the mean ± SEM from three biologically independent experiments performed in triplicate. And data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test. WT data in Fig. 2i–k are from the same experimental replicate set.
