ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

• Published in: The Journal of cell biology 2020-07, Vol.219 (7)
• Main Authors: Shi, Xiaoshan, Yokom, Adam L, Wang, Chunxin, Young, Lindsey N, Youle, Richard J, Hurley, James H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Autophagy - genetics; Autophagy-Related Protein-1 Homolog - chemistry; Autophagy-Related Protein-1 Homolog - genetics; Autophagy-Related Protein-1 Homolog - metabolism; Autophagy-Related Proteins - chemistry; Autophagy-Related Proteins - genetics; Autophagy-Related Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Binding Sites; Cell death and autophagy; Cloning, Molecular; Cryoelectron Microscopy; Deuterium Exchange Measurement; Gene Expression; Gene Expression Regulation; Genetic Vectors - chemistry; Genetic Vectors - metabolism; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins - chemistry; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Mutation; Protein Binding; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases - chemistry; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Structural Biology; Vesicular Transport Proteins - chemistry; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.201911047

The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.