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Structural basis for FLCN RagC GAP activation in MiT-TFE substrate-selective mTORC1 regulation

Jansen, Rachel M., Peruzzo, Roberta, Fromm, Simon A., Yokom, Adam L., Zoncu, Roberto, Hurley, James H.·2022·Science Advances
DOI 10.1126/sciadv.add2926PMC PMC9473554
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Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and catabolism in response to nutrients through phosphorylation of key substrates. The tumor suppressor folliculin (FLCN) is a RagC/D guanosine triphosphatase (GTPase)–activating protein (GAP) that regulates mTORC1 phosphorylation of MiT-TFE transcription factors, controlling lysosome biogenesis and autophagy. We determined the cryo–electron microscopy structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagA GDP :RagC GDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex structure, FLCN reorients by 90°, breaks contact with RagA, and makes previously unseen contacts with RagC that position its Arg 164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity and led to nuclear retention of TFE3, with no effect on mTORC1 substrates S6K or 4E-BP1. The structure provides a basis for regulation of an mTORC1 substrate-specific pathway and a roadmap to discover MiT-TFE family selective mTORC1 antagonists.

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Fig. 1. Active FLCN complex formation. (A) Construct design for the SLC38A9NT-FNIP2 fusion construct. (B) AFC complex formation workflow. (C) Size exclusion profile for AFC. Fraction containing all components of complex is labeled with a black arrow and visualized on adjacent gel.
Fig. 1. Active FLCN complex formation. (A) Construct design for the SLC38A9NT-FNIP2 fusion construct. (B) AFC complex formation workflow. (C) Size exclusion profile for AFC. Fraction containing all components of complex is labeled with a black arrow and visualized on adjacent gel.CC BY-NC · PubMed Central
Fig. 2. Cryo-EM structure of the active FLCN complex. (A) Domain organization for components of AFC. (B) Composite map for complex. Focused map of interface was combined with the overall map to produce a composite map containing the highest-resolution information for each component. (C) Reconstructed model for AFC with overall dimensions of the side view of the complex. Dotted regions in the model represent unstructured regions in FLCN connecting the two domains. Red spheres indicated locations of mutations in FLCN F10D and FNIP2 V146E.
Fig. 2. Cryo-EM structure of the active FLCN complex. (A) Domain organization for components of AFC. (B) Composite map for complex. Focused map of interface was combined with the overall map to produce a composite map containing the highest-resolution information for each component. (C) Reconstructed model for AFC with overall dimensions of the side view of the complex. Dotted regions in the model represent unstructured regions in FLCN connecting the two domains. Red spheres indicated locations of mutations in FLCN F10D and FNIP2 V146E.CC BY-NC · PubMed Central
Fig. 3. Specificity of FLCN-RagC GAP interaction in AFC. (A) Nucleotide-binding pocket of RagC (blue) in AFC containing the XDP-BeF3 nucleotide analog. The location of FLCN (orange) arg finger is indicated. Hydrogen bonds between nucleotide and RagC residues (blue sticks) in the surrounding pocket are represented by dashed lines (green). (B) Surface representation of RagC binding site with FLCN arg finger (orange). RagC is colored on the basis of electrostatic potential. (C) RagC (blue) FLCN (orange) interface in AFC. Loop residues 10 to 16 in FLCN are indicated as sticks. (D) RagA (purple) FLCN interface modeled with GTP-bound RagA (Protein Data Bank: 6S6D). Residues in the FLCN loop that clash with RagA are shown in red.
Fig. 3. Specificity of FLCN-RagC GAP interaction in AFC. (A) Nucleotide-binding pocket of RagC (blue) in AFC containing the XDP-BeF3 nucleotide analog. The location of FLCN (orange) arg finger is indicated. Hydrogen bonds between nucleotide and RagC residues (blue sticks) in the surrounding pocket are represented by dashed lines (green). (B) Surface representation of RagC binding site with FLCN arg finger (orange). RagC is colored on the basis of electrostatic potential. (C) RagC (blue) FLCN (orange) interface in AFC. Loop residues 10 to 16 in FLCN are indicated as sticks. (D) RagA (purple) FLCN interface modeled with GTP-bound RagA (Protein Data Bank: 6S6D). Residues in the FLCN loop that clash with RagA are shown in red.CC BY-NC · PubMed Central
Fig. 4. Hydrophobic residues in the FLCN:RagC interface necessary for GAP activity. (A) RagC (blue) and the FLCN (orange):FNIP2 (green) interface in AFC. FLCN:FNIP2 residues mutated at the interface indicated (red). (B) Tryptophan fluorescence xanthosine triphosphatase (XTPase) assay with FLCN:FNIP2 mutants. FLCNWT:FNIP2 (gray), FLCNF10D:FNIP2 (orange), or FLCN:FNIP2V146E (green) was incubated with RagAGDP:RagCXTP. Plotted are means ± SEM. n = 3 replicates. (C) Immunofluorescence images of human embryonic kidney (HEK) 293T cells stably expressing short hairpin RNAs targeting the indicated genes and FLCN rescue constructs. Cells were starved for amino acids (−AA) or restimulated with complete Dulbecco’s modified essential medium (+AA). (D) Quantification of TFE3 nuclear localization for immunofluorescence images and the positive control FLCN164 identified previously (8) shown in fig. S8 (means ± SD, n = 10 fields). ****P < 0.0001. (E) Fluorescence images of HEK293A cells expressing the indicated FLCN:eGFP construct along with HA-FNIP2 and the lysosomal marker Lysosomal associated membrane protein-2 (LAMP2):RFP. Cells were starved for amino acids (−AA) or starved and then restimulated with amino acids for 15 min (+AA). (F) Quantification of the lysosomal localization of overexpressed FLCN for immunofluorescence images (means ± SD, n = 15 cells). **P < 0.01. (G) Immunofluorescence images of HEK293T cells stably overexpressing the FNIP2WT-eGFP-V5 or FNIP2V146E-eGFP-V5 constructs. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (H) Quantification of TFE3 nuclear localization for immunofluorescence images (means ± SD, n = 10 fields). ****P < 0.0001. (I) Immunofluorescence images of HEK293T cells stably overexpressing the indicated FNIP2-eGFP-V5 constructs. LAMP2 was used as a lysosomal marker. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (J) Quantification of the lysosomal localization of overexpressed FNIP2 for immunofluorescence images (means ± SD, n = 15 cells). ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.
Fig. 4. Hydrophobic residues in the FLCN:RagC interface necessary for GAP activity. (A) RagC (blue) and the FLCN (orange):FNIP2 (green) interface in AFC. FLCN:FNIP2 residues mutated at the interface indicated (red). (B) Tryptophan fluorescence xanthosine triphosphatase (XTPase) assay with FLCN:FNIP2 mutants. FLCNWT:FNIP2 (gray), FLCNF10D:FNIP2 (orange), or FLCN:FNIP2V146E (green) was incubated with RagAGDP:RagCXTP. Plotted are means ± SEM. n = 3 replicates. (C) Immunofluorescence images of human embryonic kidney (HEK) 293T cells stably expressing short hairpin RNAs targeting the indicated genes and FLCN rescue constructs. Cells were starved for amino acids (−AA) or restimulated with complete Dulbecco’s modified essential medium (+AA). (D) Quantification of TFE3 nuclear localization for immunofluorescence images and the positive control FLCN164 identified previously (8) shown in fig. S8 (means ± SD, n = 10 fields). ****P < 0.0001. (E) Fluorescence images of HEK293A cells expressing the indicated FLCN:eGFP construct along with HA-FNIP2 and the lysosomal marker Lysosomal associated membrane protein-2 (LAMP2):RFP. Cells were starved for amino acids (−AA) or starved and then restimulated with amino acids for 15 min (+AA). (F) Quantification of the lysosomal localization of overexpressed FLCN for immunofluorescence images (means ± SD, n = 15 cells). **P < 0.01. (G) Immunofluorescence images of HEK293T cells stably overexpressing the FNIP2WT-eGFP-V5 or FNIP2V146E-eGFP-V5 constructs. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (H) Quantification of TFE3 nuclear localization for immunofluorescence images (means ± SD, n = 10 fields). ****P < 0.0001. (I) Immunofluorescence images of HEK293T cells stably overexpressing the indicated FNIP2-eGFP-V5 constructs. LAMP2 was used as a lysosomal marker. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (J) Quantification of the lysosomal localization of overexpressed FNIP2 for immunofluorescence images (means ± SD, n = 15 cells). ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.CC BY-NC · PubMed Central

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Metadata

Authors
Jansen, R. M., Peruzzo, R., Fromm, S. A., Yokom, A. L., Zoncu, R., & Hurley, J. H.
Affiliations
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2022-09-16
Volume
8
Issue
37
ISSN
2375-2548
DOI
10.1126/sciadv.add2926
PMC
PMC9473554
Cited by
42
References
6
Language
en

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