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Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9

Nature Structural & Molecular Biology volume 27, pages 1017–1023 (2020)Cite this article

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Abstract

The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

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Fig. 1: SLC38A9NT triggers FLCN GAP activity and disassembles the LFC.
Fig. 2: Cryo-EM structure of the pre-GAP complex.
Fig. 3: HDX-MS difference of SLC38A9NT reveals pre-GAP complex dynamics.
Fig. 4: Rag GTPases are trapped in the inactive conformation in the post-GAP complex.
Fig. 5: SLC38A9NT blocks spontaneous nucleotide exchange in RagA.

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Data availability

EM density maps have been deposited in the EMDB with accession codes EMD-21686 (pre-GAP complex) and EMD-21687 (post-GAP complex). Atomic coordinates have been deposited in the PDB with accession codes 6WJ2 (pre-GAP complex) and 6WJ3 (post-GAP complex). Source data are provided with this paper.

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Acknowledgements

We thank R. Zoncu for comments on the manuscript, C. Hecksel for assistance in pre-GAP complex cryo-EM data acquisition, which was performed at the Stanford-SLAC Cryo-EM Center (S2C2) supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129541), and D. Toso, J. Remis and P. Tobias for assistance in post-GAP complex cryo-EM data acquisition. This work was supported by NIH R01GM111730 (J.H.H.), an EMBO Long-Term Fellowship (S.A.F.) and a University of California Cancer Research Coordinating Committee Predoctoral Fellowship (R.E.L.).

Author information

Author notes

  1. Rosalie E. Lawrence

    Present address: Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Simon A. Fromm, Rosalie E. Lawrence & James H. Hurley

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA

    Simon A. Fromm, Rosalie E. Lawrence & James H. Hurley

  3. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    James H. Hurley

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Contributions

S.A.F. designed and carried out all experiments and carried out all data analysis. R.E.L. performed initial GEF and LFC disassembly experiments. S.A.F. and J.H.H. conceptualized the project and wrote the first manuscript draft. All authors contributed to editing the manuscript.

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Correspondence to James H. Hurley.

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Competing interests

J.H.H. is a scientific founder and receives research funding from Casma Therapeutics.

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Peer review information Peer reviewer reports are available. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Pre−GAP complex cryo-EM structure determination.

a, Exemplary raw cryo-EM micrograph at −2.2 μm defocus. Scale bar 50 nm. b, Power spectrum of micrograph shown in a with CTF estimation. c, Exemplary 2D class averages. Scale bar 150 Å. d, Cryo-EM data processing workflow. Used software is indicated with italic font. Red asterisk indicates the map used for model building to subsequently generate simulated maps w/o SLC38A9. e, Particle orientation distribution of the final particle set. f, Fourier shell correlation (FSC) of the final 3D reconstruction. g-h, Overlay of the final density map with the masks used during refinement (g, blue, transparent), FSC calculation (h, pink, transparent). i, Both masks from g and h overlaid with the final density map.

Extended Data Fig. 2 Pre−GAP complex atomic coordinate building and refinement.

a, Overlay of half-map (green) and map-model (purple) FSC to assess map to model agreement. b, Overlay of FSC work (blue) and FSC test (yellow) of the cross-validation test to assess overfitting. The refinement target resolution is indicated by a vertical dashed line. c, Final model composition and chain assignment. Parts not resolved by the cryo-EM density are represented by thin black lines. Red lines indicate regions where side chains are truncated to alanine. d, Model fit in the cryo-EM density (mesh) of selected regions. The threshold level used to display the density in UCSF Chimera is given in parentheses.

Extended Data Fig. 3 HDX-MS analysis of SLC38A9NT in isolation and bound to Rags.

a, Deuterium uptake and peptide coverage (grey lines) of SLC38A9NT in complex with inactive Rags (left) or in isolation (right) at 6, 60, 600 and 60,000 s exchange time. b, HDX difference plots of SLC38A9NT in complex with inactive Rags and in isolation at 60 (left), 600 (middle) and 60,000 s (right) exchange time. Plotted are the mean±SD of technical replicates (n = 3). c, Individual SLC38A9NT MS peptide spectra of selected peptides in isolation (black) and in complex with inactive Rags (red). Undeuterated reference spectra are shown at the top. Data for graphs in b are available as source data online.

Source data

Extended Data Fig. 4 Post-GAP complex cryo-EM structure determination.

a, Exemplary raw cryo-EM micrograph at −2.2 μm defocus. Scale bar 50 nm. b, Power spectrum of micrograph shown in a with CTF estimation. c, Exemplary 2D class averages. Scale bar 150 Å. d, Cryo-EM data processing workflow. Used software is indicated with italic font. e, Particle orientation distribution of the final particle set. f, Fourier shell correlation (FSC) of the final 3D reconstruction.

Extended Data Fig. 5 Post-GAP complex atomic coordinate building and refinement.

a, Overlay of half-map (green) and map-model (purple) FSC to assess map to model agreement. b, Overlay of FSC work (blue) and FSC test (yellow) of the cross-validation test to assess overfitting. The refinement target resolution is indicated by a vertical dashed line. c, Model fit in the cryo-EM density (mesh) of selected regions. The threshold level used to display the density in UCSF Chimera is given in parentheses.

Extended Data Fig. 6 Overview of Rag GTPase structures in different states.

a–g, Top (middle) and side view (right) of published Rag GTPase structures in surface representation (cyan, RagA G domain; blue, RagC G domain; pink, GTP or GTP analogue; red, GDP or GDP analogue; yellow, RagA or RagC switch I region; grey, RagA or RagC C-terminal roadblock domain). The dashed line connects the Cα atoms of RagA Trp165 and RagC Tyr221 representing the width of the G domain cleft. The two vertical solid lines represent the RagA Trp165 and RagC Tyr221 Cα position in a. PDB codes, experimental method, de facto nucleotide state and Rag binding partners (if any) are summarized on the left.

Extended Data Fig. 7 Tryptophan fluorescence-based RagC XTPase assay.

a, Intrinsic tryptophan fluorescence based RagC XTPase assay of Ragulator-RagAGDP:RagCXTP in the absence (blue) and presence (orange) of SLC38A9NT. Tryptophan fluorescence of a Ragulator-RagAGTP:RagCXTP substrate used in the same assay serves as a positive control (green). Plotted is the mean ± SD mantGDP fluorescence at each time point of one experiment performed in quadruplicates (n = 4). The experiment has been performed twice with similar results. norm., normalized. b, Top view of the pre-GAP complex structure illustrating the SLC38A9NT-RagC switch I and GDP (RagA, red) interaction. SLC38A9 (yellow) and RagC switch I (blue) are displayed in surface representation. Data for graph in a is available as source data online.

Source data

Supplementary information

Source data

Source Data Fig. 1

Source data for SEC and statistical source data for HPLC-based XTPase assay.

Source Data Fig. 1

Unprocessed SDS gels.

Source Data Fig. 3

Statistical source data for HDX-MS experiment.

Source Data Fig. 4

Source data for SEC.

Source Data Fig. 4

Unprocessed SDS gels.

Source Data Fig. 5

Statistical source data for HPLC and mantGDP-based GEF assays.

Source Data Extended Data Fig. 3

Statistical source data for HDX-MS experiment.

Source Data Extended Data Fig. 7

Statistical source data for Trp fluorescence–based GEF assay.

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Fromm, S.A., Lawrence, R.E. & Hurley, J.H. Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9. Nat Struct Mol Biol 27, 1017–1023 (2020). https://doi.org/10.1038/s41594-020-0490-9

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