Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex

Abstract

Summary The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners.

Atg101: Not Just an Accessory Subunit in the Autophagy-initiation Complex

“…It is likely that the closed conformation of Atg13 HORMA is stabilized by O-Mad2-like Atg101 just as C-Mad2 is stabilized by O-Mad2. Soon after the fission yeast structure was reported, two groups independently reported the crystal structure of human Atg101 alone or as a complex with human Atg13 HORMA (Michel et al, 2015;Qi et al, 2015). These studies confirmed that human Atg101 also has the OMad2-like HORMA fold and stabilizes human Atg13 HORMA in a similar manner (Fig.…”

“…Similarly, the crystal structure of human Atg101 complexed with Atg13 shows that the equivalent loop region in human Atg101 interacts with a hydrophobic pocket in Atg13 and the hydrophobic compound, benzamidine, using Trp110 and Phe112 (Fig. 3F) (Qi et al, 2015). These interactions are most likely an artifact of crystallization but they indicate the potential of this loop region to interact with hydrophobic pockets in other proteins.…”

“…The cap locks the C-terminal segment of LtAtg13 through extensive interactions, which then seems to stabilize the C-Mad2-like conformation of LtAtg13 without Atg101, although the manner of stabilization by the cap is different from that by Atg101: Atg101 interacts with the C-terminal β-strands, whereas the cap interacts with the loop region preceding the C-terminal β-strands. The cap is not observed in the structures of fission yeast and human Atg13 HORMA (Michel et al, 2015;Qi et al, 2015;Suzuki et al, 2015). Thus, the evidence suggest that Atg13 with a cap insertion is stable on its own whereas that lacking a cap is intrinsically unstable and requires Atg101 for stabilization.…”

“…The interface between Atg101 and Atg13 HORMA is also mostly conserved between fission yeast and human and point mutation of Phe29 and His30 in SpAtg101 and their equivalent Leu30 and His31 in human Atg101 equally impaired the interaction with Atg13 HORMA (Suzuki et al, 2015). One exception is the salt bridge between Atg13 Arg133 and Atg101 Asp54 in the human complex-it is absent from the fission yeast complex but seems to be highly conserved among animals (Qi et al, 2015).…”

“…The crystal structure of the Nterminal domain of Atg13 from yeasts and human revealed that it has a HORMA fold (described below and Fig. 3) (Jao et al, 2013;Michel et al, 2015;Qi et al, 2015;Suzuki et al, 2015). The crystal structure of Atg13 MIM was determined as a complex with the C-terminal domain of C. Domain organization of Atg17, Atg11 and FIP200.…”