Structural basis for the ARF GAP activity and specificity of the C9orf72 complex

Ming-Yuan Su,James H Hurley,Simon A Fromm,Daniel B Toso,Jonathan Remis

doi:10.1038/s41467-021-24081-0

Abstract

Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required ~10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP. The structure also provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway.

Highlights

Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function
An ARF1 construct with the N-terminal myristoylated amphipathic helix removed was used in all experiments, as this region was previously found to be unnecessary for its competence as a GTPase activating protein (GAP) substrate[21]
The concentration of ARF1 was fixed at 30 μM, and treated with 0, 0.3, 0.6, and 3-μM C9orf[72] complex for 15 min at 37 °C, and nucleotide products were monitored by high pressure liquid chromatogram (HPLC) analysis

Summary

Introduction

Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. The structure provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway. C9orf[72] exists in cells as a stable heterotrimer with Smith–Magenis chromosome regions 8 (SMCR8) and WD repeat-containing protein 41 (WDR41)[9,10,12,13,14,15] Both C9orf[72] and its cellular binding partner SMCR8 are longin and DENN domain (differentially expressed in normal and neoplastic cells) containing proteins[19]. RagC in the mTORC1 signaling pathway, with the putative catalytic Arg residue residing in the FLCN subunit This suggested that C9orf72:SMCR8 might be a GAP for some small GTPase[20,21]. In the absence of a structure determination for the active state of any of these complexes, there is not yet a consensus in the field with respect to the Arg finger mechanism[24]

Methods

Results

Conclusion