Structural insights into cholesterol sensing by the LYCHOS-mTORC1 pathway

Abstract

The lysosomal cholesterol sensor LYCHOS regulates mTORC1 signaling by coupling cholesterol sensing to GATOR1-Rag GTPase modulation, yet its structural mechanisms remain unclear. Here we report six cryo-electron microscopy structures of human LYCHOS, depicting five distinct states. These are categorized into a contracted state when complexed with a sufficient amount of the cholesterol analogue cholesteryl hemisuccinate (CHS), and an expanded state when CHS is deficient. The structure forms a homodimer, within each monomer the transmembrane region is divided into a permease-like domain (PLD) and a GPCR-like domain (GLD) with two clearly defined adjacent cholesterol binding sites between them. Cholesterol binding induces a translation of GLD towards PLD and exposes the cytosolic extension of transmembrane 15, which interacts with GATOR1. Our results elucidate the structural mechanism of cholesterol sensing by the mTORC1 pathway, providing a structural basis for developing inhibitors that selectively target mTORC1 pathway by blocking LYCHOS in its expanded state.

Fig. 1. Cryo-EM structures of human LYCHOS.

a MST experiments showing that our purified LYCHOS with CHS has a similar binding affinity for GATOR1 (Kd = 7.6 μM) compared with LED (Kd = 12 μM). Values are shown as the mean ± SD from three biologically independent experiments (n = 3). b–d Cryo-EM maps of lipids bound expanded LYCHOS_{WT_E}, CHS bound contracted LYCHOS_{WT_C} and CHS bound contracted LYCHOS_{Y57A_C}. The two protomers are colored in slate blue and light coral, and TM15 is shown in deep sky blue. The positions of TM15 of LYCHOS_{WT_E} expanded conformation and LYCHOS_{Y57A_C} contracted conformation are indicated by dashed line and solid line. CHS are shown in green, and corresponding cryo-EM map are shown as insets. Other lipid molecules are shown in light gray. e Schematic of the LYCHOS secondary structure features. Regions that are not visible are shown in dashed lines. Densities for α0 are very poor and also shown in dashed cylinders. Residues that affect LYCHOS function in previous studies are shown as red dots. Resolved boundary residues are shown in blue dots. Scaffold domain, transporter domain, GLD, LED, and DEP are shown as gold, purple, deep sky blue, blue and green, respectively. f Structural model of LYCHOS_{Y57A_C} shown in cartoon representation. Key domains are colored in protomer A as in (e). TM15 in protomer B is shown in deep sky blue.

Fig. 2. Cholesterol binding sites.

a, b Electrostatic potential surfaces (a) and cartoon representation (b) of protomer A of the LYCHOS_{WT_C} structure. CHS molecules are shown as sticks. c Details of the cholesterol binding site CBS1 of LYCHOS_{WT_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.

Fig. 3. LYCHOS adopts an expanded conformation without sufficient cholesterol.

a Cartoon representation of LYCHOS_{WT_C} (cyan) and LYCHOS_{WT_E} (yellow), superimposed based on the PLD domain of chain A in LYCHOS_{WT_C} and chain B in LYCHOS_{WT_E}. b Conformational displacements of the GLD relative to the PLD between LYCHOS_{WT_C} (cyan) and LYCHOS_{WT_E} (yellow). Cα atoms of residues used to measure displacements are shown in sticks and labeled. c Schematic representation of the displacement of the four residues highlighted in b. Dashed lines indicate distances between the Cα atoms within each state. Solid arrows indicate the absolute distance between Cα atoms of each residue between LYCHOS_{WT_E} and LYCHOS_{WT_C}. d, e The distance between Tyr551 and bilayer phospholipids is larger in LYCHOS_{WT_C} when compared with LYCHOS_{WT_E}. The bilayer phospholipids are shown in stick. Tyr551 is shown in stick and colored in magenta. The states were revealed by 1 μs molecular dynamics simulation. f Schematic of the FlAsH-BRET assay. Nluc inserted at intracellular loops (ICL2 or ICL5) of LYCHOS (transporter-like domain) and the FlAsH motif (CCPGCC) incorporated in ICL7, ICL8 or C-terminus of LYCHOS (GPCR-like domain). LYCHOS_{WT_E} (gray) and LYCHOS_{WT_C} (green) are shown from cytosolic view. g Detailed description of the Nluc and FlAsH motif incorporation sites at the ICLs of LYCHOS. h, i The maximal response of six intramolecular fluorescent arsenical hairpin (FlAsH)–bioluminescence resonance energy transfer biosensors of LYCHOS in response to cholesterol stimulation; The values are reported as the mean ± SEM of three biologically independent experiments (n = 3). j, k Concentration-dependent curves of LYCHOS FlAsH-BRET sensors in response to cholesterol (n  =  3 biologically independent experiments). l Cartoon representation of LYCHOS_{WT_C} (cyan) and LYCHOS_{Y57A_C} (slate), superimposed based on the PLD domain. m Conformational displacements of the LYCHOS_{WT_C} (cyan) bound CHS molecules (cyan) and LYCHOS_{Y57A_C} (slate) bound CHS molecules (green) relative to the PLD. Solid arrows indicate the absolute distance between carboxylate oxygen atoms of CHS molecules between LYCHOS_{WT_C} and LYCHOS_{Y57A_C}. n Superimposition of the LYCHOS_{Y57A_C} structure with the map of LYCHOS_{Y57A/R61A_E}. The solid arrow indicates the translation of GLD.

Fig. 4. Lipids reside at the dimerization interface of LYCHOS.

a Cryo-EM maps of LYCHOS_PLD homodimer bound with lipids. The two protomers are colored in slate blue and light coral. Lipid molecules are shown in light gray. b Cryo-EM maps of LYCHOS_{Y57A_C} bound with PS. The two protomers are colored in slate blue and light coral. Two lipid molecules are colored in orange and green, respectively. c Cryo-EM density maps of PS in the LYCHOS_{Y57A_C} state are shown in blue meshes. d Details of the interaction between lipids and LYCHOS. PS and CHS molecules are shown as sticks. Residues interacting with lipids are shown in sticks. e Superimposition of the LYCHOS_{Y57A_C} structure with the map of LYCHOS_{Y57A_TM6_E}. The two protomers are colored in slate blue and light coral for LYCHOS_{Y57A_C} and lipids are shown in sticks. The lipids observed in the canonical dimer interface of LYCHOS_{Y57A_C} could not be seen in LYCHOS_{Y57A_TM6_E}.

Fig. 5. Proposed mechanism of LYCHOS.

Up: Under low cholesterol, LYCHOS is in an expanded state in the absence of cholesterol. GATOR1 promotes GTP hydrolysis of RagA/B, maintaining mTORC1 in the cytosol and inactive. Down: When cholesterol is sufficient, binding of cholesterol induces the contraction of the GLD and the descending of TM15, makes the LED suitable for interaction with GATOR1, thus facilitating the GTP-loaded state of RagA/B that recruits mTORC1 to the lysosome and activate it.