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

Area of Science:

• Structural Biology
• Neurogenetics
• Molecular Biology

Background:

• C9orf72 mutations are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).
• The C9orf72 protein complex, including SMCR8 and WDR41, exhibits GTPase activating protein (GAP) activity towards ARF and RAB proteins.
• Understanding the molecular mechanisms of C9orf72 function is crucial for elucidating disease pathogenesis.

Purpose of the Study:

• To determine the cryo-electron microscopy (cryo-EM) structure of the C9orf72:SMCR8:WDR41 complex bound to ARF1.
• To elucidate the structural basis of the GTPase activating protein (GAP) activity of the C9orf72 complex.
• To investigate the functional impact of mutations in interfacial residues.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of the C9orf72 complex bound to ARF1-GDP-BeF3-.
• Biochemical assays to assess GTPase activating protein (GAP) activity.
• Site-directed mutagenesis to probe the function of interfacial residues.

Main Results:

• The cryo-EM structure reveals how the SMCR8 and C9orf72 longin domains form the binding pocket for ARF1.
• Specific residues in the C9orf72 and SMCR8 longin domains are critical for positioning ARF1 and its catalytic site.
• Mutations in interfacial residues significantly reduced or abolished GAP activity towards ARF1.
• The C9orf72 complex demonstrates a specific and potent GAP activity towards ARF1, with lower activity towards RAB8A.

Conclusions:

• The C9orf72 complex functions specifically as an ARF GAP, providing a molecular basis for its role in neurodegeneration.
• The determined structure offers insights into the mechanism of longin domain GAPs, including those regulating the mTORC1 pathway.
• This study provides a structural foundation for understanding how C9orf72 mutations lead to ALS and FTD.