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

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 electron microscopy (EM) and multiangle light scattering showed that FIP200 is a dimer whilst a single molecule each of the other subunits is present. The FIP200 NTD 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 structure similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy initiating TBK1 kinase and the ULK1 kinase complex. Summary The human ULK complex consists of ULK1/2, FIP200, ATG13, and ATG101. We found that the FIP200 N-terminal domain is a C-shaped dimer that binds directly to a single ATG13 molecule and serves as the organizing hub of the complex.