Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD

Abstract

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4,5,6. Here we determine the structure of the C9orf72–SMCR8–WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a β-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72–SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72–SMCR8–WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.

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Fig. 1: Cryo-EM structure of the C9orf72–SMCR8–WDR41 complex.
Fig. 2: HDX-MS of C9orf72–SMCR8 in the absence of WDR41.
Fig. 3: SMCR8 mutants do not localize on lysosomes.
Fig. 4: C9orf72–SMCR8 is a GAP for ARF proteins.

Data availability

The electron microscopy density map has been deposited in the Electron Microscopy Data Bank with accession number EMD-21048. Atomic coordinates for C9orf72–SMCR8–WDR41 have been deposited in the PDB with accession number 6V4USource data are provided with this paper.

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Acknowledgements

We thank D. Toso, K. L. Morris, V. Kasinath and P. Tobias for cryo-EM advice and support; X. Shi for HDX support; C. Behrends and G. Stjepanovic for suggestions and contributions to the early stages of the project; R. Lawrence and M. Lehmer for cell imaging advice; and X. Ren for assistance with cloning. This work was supported by NIH grants R01GM111730 (J.H.H.) and R01GM130995 (R.Z.), Department of Defense Peer Reviewed Medical Research Program Discovery Award W81XWH2010086 (J.H.H.), the Bakar Fellows program (J.H.H.), the Pew-Stewart Scholarship for Cancer Research and Damon Runyon-Rachleff Innovation Award (R.Z.), a postdoctoral fellowship from the Association for Frontotemporal Degeneration (M.-Y.S.) and an EMBO Long-Term Fellowship (S.A.F.).

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Authors

Contributions

M.-Y.S. designed and carried out all experiments, except for GEF assays and lysosome colocalization experiments, and performed all data analysis. S.A.F. performed GEF assays. R.Z. carried out lysosome colocalization experiments. M.-Y.S. and J.H.H. conceptualized the project and wrote the first draft. All authors contributed to the editing of the manuscript.

Corresponding author

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. R.Z. is a co-founder and stockholder in Frontier Medicines Corp.

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Peer review information Nature thanks Aaron Burberry, Sjors Scheres and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Purification of the C9orf72–SMCR8–WDR41 and C9orf72–SMCR8 complex, as well as the HDX data for the trimer.

a, The Superose-6 gel filtration elution profile for the C9orf72–SMCR8–WDR41 complex. b, The Superose-6 gel filtration elution profile for the C9orf72–SMCR8 complex. mAU, milli-absorbance units. c, The purified full-length C9orf72–SMCR8–WDR41 and C9orf72–SMCR8 complexes were analysed by SDS–PAGE. The proteins were purified at least five times with similar results (ac). df, Deuterium uptake data for the C9orf72–SMCR8–WDR41 complex at the 0.5-s time point, with error bars from triplicate technical measurements. Peptides with more than 50% deuterium uptake are the flexible regions. Y axis represents the average per cent deuteration. X axis demonstrates the midpoint of a single peptic peptide. Source data

Extended Data Fig. 2 Cryo-EM data processing.

a, A representative cryo-EM micrograph of the C9orf72–SMCR8–WDR41 complex. b, Representative 2D classes. c, Orientation distribution of the aligned C9orf72–SMCR8–WDR41 particles. d, Image processing procedure.

Extended Data Fig. 3 Resolution estimation of the cryo-EM map, as well as model building and validation.

a, Comparison between FSC curves. b, Refined coordinate model fit of the indicated region in the cryo-EM density. c, Refinement and map-versus-model FSC. d, Cross-validation of test FSC curves to assess overfitting. The refinement target resolution (4.2 Å) is indicated. e. Different views of the final reconstruction. Source data

Extended Data Fig. 4 Deuterium uptake of C9orf72–SMCR8–WDR41.

HDX-MS data are shown in heat map format, in which peptides are represented by rectangular strips above the protein sequence. Absolute deuterium uptake after 0.5, 5, 50, 500 and 50,000 s are indicated by a colour gradient below the protein sequence.

Extended Data Fig. 5 Deuterium uptake of C9orf72–SMCR8 complex.

HDX-MS data are shown in heat map format, in which peptides are represented by rectangular strips above the protein sequence. Absolute deuterium uptake after 0.5, 5, 50, 500 and 50,000 s are indicated by a colour gradient below the protein sequence.

Extended Data Fig. 6 Mapping the protected region from HDX results onto the SMCR8–C9orf72–WDR41 structure.

a, The HDX uptake difference at 0.5 s was mapped on C9orf72–SMCR8. bd, Close-up view of M1 (b) and M2–M5 (c) regions of SMCR8, and the M1 region of C9orf72 (d). e, Enlarged view of the WDR41 residues in the SMCR8–WDR41 interface.

Extended Data Fig. 7 Co-expression and pulldown validation of C9orf72–SMCR8 and SMCR8–WDR41 interface.

a, Close-up view of the residues that mediate the DENN– DENN dimerization between C9orf72 and SMCR8. b, Co-expression and pulldown experiment of Strep-tagged SMCR8 with GST–C9orf72 mutants and WDR41. c, Pulldown experiment of GST–WDR41 mutants with C9orf72–SMCR8. The pulldown experiments were carried out at least twice with similar results (b, c).

Extended Data Fig. 8 Structural comparison between C9orf72–SMCR8 and FNIP2–FLCN.

Left, structural alignment of full-length FNIP2–FLCN and C9orf72–SMCR8. Right, comparison between the longin dimers (top), DENN dimers (middle) and SMCR8 DENN with C9orf72 DENN domain (bottom).

Extended Data Fig. 9 GTPase assay for different small GTPases with C9orf72–SMCR8–WDR41.

a, SDS–PAGE of GAP protein complex (top) and GTPase proteins (bottom) used in the experiments. b, Tryptophan fluorescence GTPase signal was measured for purified RAGA–RAGC, ARL8A, ARL8B, RAB1A and RAB7A before and after addition of C9orf72–SMCR8–WDR41. The fluorescence signal upon GAP addition was normalized to 1 for each experiment. The experiments were carried out in triplicate and one representative plot is plotted. c, Tryptophan fluorescence GTPase signal was measured for purified ARF6, ARF(Q67L) or ARF5, before and after addition of C9orf72–SMCR8–WDR41, C9orf72–SMCR8(R147A)–WDR41 or C9orf72–SMCR8. d, HPLC-based GTPase assay with ARF6, ARF5, RAB1A, RAB7A, ARL8A, ARL8B and RAGA–RAGC proteins in the absence and addition of C9orf72–SMCR8–WDR41 complex, as indicated. The experiments were carried out in triplicate and one representative plot is shown. All experiments were carried out at least three times independently with similar results (ad). Source data

Extended Data Fig. 10 HPLC-based GTPase assay with ARF1 or ARF1(Q71L) proteins.

Assays were performed in the absence and addition of GAP complex, as indicated. The experiments were carried out in triplicate and one representative plot is shown. All experiments were carried out at least three times independently with similar results.

Extended Data Fig. 11 GEF assay for different small GTPases with C9orf72–SMCR8–WDR41.

a, SDS–PAGE of C9orf72–SMCR8–WDR41 complex and GTPase proteins used in the experiments. b, GEF assay with mantGDP-reloaded RAB1A, RAB5A, RAB7A, RAB8A and RAB39B proteins in the absence and addition of C9orf72–SMCR8–WDR41 complex, as indicated. RAB5A treated with RABEX5 was used as a positive-control reaction. Data were baseline-subtracted and normalized to the signal immediately after GTP addition, which also is the 0-s time point in the plots. Plots are mean ± s.d. of each technical triplicate experiment. All experiments were carried out at least twice independently with similar results (a, b). c, Structural alignment of C9orf72–SMCR8–WDR41 with DENND1B–RAB35 (PDB 3TW8). Source data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

This file contains Supplementary Fig.1 presenting the uncropped gels for Fig. 2 and Extended Data Figs. 1, 7, 9, and 11.

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Supplementary Data

This file contains Supplementary Dataset 1 presenting the HDX data summary.

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Su, M., Fromm, S.A., Zoncu, R. et al. Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD. Nature 585, 251–255 (2020). https://doi.org/10.1038/s41586-020-2633-x

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