ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer
Posted on: 17 December 2019 , updated on: 18 December 2019
Preprint posted on 12 November 2019
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201911047
The FIP200 N-terminal domain orchestrates assembly of the autophagy-inducing ULK complex
Selected by Sandra Malmgren Hill
Categories: biochemistry, biophysics, cell biology, molecular biology
Context
The ULK complex is one of the most upstream and core complexes for regulating the cellular degradation and recycling process known as autophagy. Upon cellular cues, the ULK complex relocalizes to mark the sites of autophagy induction and recruits downstream effectors that eventually lead to the formation of a double membrane autophagosome, which encloses cargo and is sent to the lysosome for destruction1.
The human ULK complex consists of the serine/threonine kinase ULK1 (or its homologue ULK2) together with the scaffolding unit FIP200, ATG13, and ATG101. Upon activation and complex assembly, ULK1 autophosphorylates itself and the members of the ULK complex, as well as a range of substrates involved in different steps of autophagy regulation.
While a lot of functional studies have been carried out to describe the function of the ULK complex in relation to autophagy, information of the structure and assembly of the ULK complex itself is lacking. The authors of this pre-print focus on the FIP200 component and evaluate the possibility of this large protein acting as a scaffold for the assembly of the ULK complex. Using advanced technologies such as Hydrogen-deuterium exchange mass spectrometry, electron microscopy (EM) and Multiangle light scattering (MALS) to map the organization and stoichiometry of the complex, they show that two molecules of FIP200 dimerize at the N-terminal domain, forming a platform for the binding of ULK1, ATG13 and ATG101.
Main findings
FIP200 is the largest protein of the ULK complex, and consists of an N-terminal domain, a coiled-coiled domain and a C-terminal CLAW domain (Fig. 1A). The CLAW domain has been shown to be involved in binding to autophagy receptors, and the structure of this domain was recently solved2. The authors of this preprint determined that the N-terminal domain of FIP200 is essential for the interaction with the other components of the ULK complex. The authors purified the N-terminal domain alone (FIP200 NTD) to determine its structure and found that it forms a C-shaped dimer, with the C-terminal part of the domain at the dimer interface (Fig. 1B). The FIP200 dimer binds directly to ATG13, and ATG13 then interacts with ATG101 by heterodimerization via the HORMA domains resident in both proteins (Fig. 1A). In immunoprecipitation experiments, the authors showed that ATG13 and ATG101 could efficiently be pulled down by FIP200 NTD, whereas ULK1 could not, indicating that ULK1 recruitment to the complex requires the pre-existence of ATG13-ATG101.
The binding of ATG13-ATG101 stabilized the C-shaped structure of the FIP200 NTD dimer, and loss of binding had a destabilizing effect on the structure as well as on FIP200 protein levels. Further investigation using Hydrogen-Deuterium exchange mapped the region of interaction between FIP200 NTD and ATG13-ATG101 to the M6 region of FIP200 (aa443-450), and the middle region (MR; aa363-460) of ATG13 (Fig. 1A). Binding of the ULK1 EAT domain to ATG13 was mapped to the C-terminal region of ATG13, in agreement with previously published data3.
Figure 1. Interactions and assembly of the ULK complex. A) FIP200, ATG13, ATG101 and ULK1 visualized in linear form from amino (N) to carboxy terminal (C), with location of protein domains annotated by residue number. Interacting domains are denoted by connecting lines.B) Overview of the suggested ULK complex structure, with complex formation orchestrated by the FIP200 NTD, with the C-terminal of FIP200 (containing the adaptor binding CLAW domain) and the N-terminal of ULK1 (containing its kinase domain) protruding away from the interaction hub.
To localize the position of the interacting components in the structure, the authors tagged individual ULK components with a Maltose-binding-protein (MBP) tag, which is a two domain 40 kDa tag that is visible under electron microscopy. This analysis demonstrated that the ATG13 MR binds in the middle of the FIP200 NTD C shape, whereas the ULK1 EAT domain is located more towards the tip of the C-shape in the presence of ATG13 and ATG101 (Fig. 1B).
From these studies the authors could conclude that the ULK1 complex is asymmetric with a nonequal subunit stoichiometry of 2:1:1:1 FIP200:ATG13:ATG101:ULK1 (Fig. 1B), where the ULK complex assembly occurs at the FIP200 NTD dimer interface, with the C-terminal of FIP200 and the N-terminal of ULK1 projecting away from the complex assembly point.
Open questions
- Since there is no evidence of impaired autophagy when expressing ULK1 components with impaired in vitro binding, it remains to be determined how complex assembly is regulated in vivo. Are there additional adapters that could facilitate complex assembly, or are the proteins able to interact via other domains than those shown to be important for binding in vitro? As FIP200 was described to have a conserved TBK1 like domain, is it possible that TBK1 is involved in this process and that TBK1 could compensate to maintain adequate autophagy induction in the absence of a functional ULK complex?
- How is the complex assembly regulated in relation to autophagy induction? Do any of the known post-translational modifications on the ULK components affect FIP200 dimerization or complex assembly? ULK1 is known to autophosphorylate itself in the EAT domain to negatively regulate autophagy4 – could this regulation be further explained by a negative effect on complex assembly? Does FIP200 binding to autophagy adaptors influence the assembly of the complex?
- The C-shape described for the FIP200 NTD differs from its functional ortholog of Atg17 which is found as an S-shaped dimer, and the question remains how the C-shape influences function of the complex. The authors hypothesize that this shape could allow for binding to ATG9 vesicles or to the edge of a growing phagophore. It will be very interesting to see if further in vitro studies using liposomes of different sizes could shed a light on this matter.
- Is it possible to make any assumptions on the timing of the ULK complex assembly?
While immunoprecipitation data showed that there was no direct interaction between FIP200 and ULK1, indicating that ATG13 is acting as a linker between the two proteins, the authors later showed that FIP200 NTD could pull down ULK1 in the presence of ATG13ΔC, which can bind FIP200 but not ULK1. Is it possible that ULK1 binds an overlapping region on FIP200-ATG13 and that these two proteins need to interact before ULK1 is recruited to the complex? Or is it possible that ULK1 binds more than one region of ATG13 and that it can still interact with ATG13ΔC?
Why I chose this preprint
Autophagy is one of the major degradative pathways in the cell, essential for the proper maintenance and recycling of cellular material. Understanding its regulation and possible ways of manipulating this process is desirable in relation to many human ailments such as neurodegenerative diseases and cancer. This preprint highlights an important gap in our knowledge and provides information on one of the core players of autophagy induction. I find that this preprint, as well as other publications from the Hurley lab, effectively bridges the gap between structural and functional biology and provides a foundation for further studies on the FIP200 regulation of ULK complex.
References
1 Lin, M. G. & Hurley, J. H. Structure and function of the ULK1 complex in autophagy. Curr Opin Cell Biol 39, 61-68, doi:10.1016/j.ceb.2016.02.010 (2016).
2 Turco, E. et al. FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates. Mol Cell 74, 330-346 e311, doi:10.1016/j.molcel.2019.01.035 (2019).
3 Chan, E. Y., Longatti, A., McKnight, N. C. & Tooze, S. A. Kinase-inactivated ULK proteins inhibit autophagy via their conserved C-terminal domains using an Atg13-independent mechanism. Molecular and cellular biology 29, 157-171, doi:10.1128/MCB.01082-08 (2009).
4 Liu, C. C. et al. Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination. Mol Cell 61, 84-97, doi:10.1016/j.molcel.2015.11.001 (2016).
doi: https://doi.org/10.1242/prelights.15858
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