Structural characterization of autophagy related protein complexes

Autophagy is a conserved and highly regulated process in eukaryotic cells that plays an important role in maintaining cellular homeostasis. During macroautophagy a double membrane grows de novo that encloses cytoplasmic cargo and after its fusion an autophagosome vesicle is formed. The autophagosome then fuses with the vacuole or lysosome, where its content is degraded. The Atg12~Atg5/Atg16 complex is an essential part of the core autophagy machinery and localizes to the outside of the growing isolation membrane. The complex also acts as an E3-like ligase for the lipidation of ubiquitin-like Atg8. The PROPPIN (β-propeller that binds polyphosphoinositides) Atg21 determines the site of Atg8 lipidation in yeast by interacting with both the coiled coil domain of Atg16 and Atg8. In my first project, I obtained first low resolution insights into the interactions of Atg21 with the coiled coil domain of Atg16. The 4.0 Å crystal structure shows that the Ashbya gossypii Atg16 coiled coil domain is at the center of the Atg21-Atg16 complex and interacts with two Kluyveromyces lactis Atg21 molecules on either site of the C-terminal ends of the coiled coil dimer. The two Atg21 molecules adopt a reversed V shape and their PI(3)P binding sites are located opposite to the Atg16 binding site. The structure thus shows how membrane bound Atg21 can bind an Atg16 dimer. However, at 4.0 Å resolution molecular details of complex interaction are not visible. I also determined crystal structure of the coiled coil domain of AgAtg16 comprising residues 43-108 at 3.4 Å resolution. Analysis of Atg21-Atg16 complex formation by analytic gel filtration revealed the importance of residues KlAtg21 R103E and AgAtg16 (70-124) D78R for binding. The structure of Atg21-Atg16 complex gives more insights into the coordination of Atg8 lipidation. The coiled coil domain of mammalian Atg16 is an effector of Golgi-resident Rab33B. In my second project, I determined the crystal structure of murine Rab33B with the Atg16L1 coiled coil domain at 3.47 Å resolution. The structure revealed that two Rab33B molecules form a complex with the diverging C-termini of one Atg16L1 dimer. Protein-protein interactions observed in the structure were confirmed by cross linking of the Rab33B(30-202)Q92L-Atg16L1(153-210) complex and analysis by mass spectrometry. Based on the structure Rab33B and Atg16L1 mutants were designed to verify the Rab33B-Atg16L1 interactions. Both in vivo and in vitro pull-down experiments showed that selected single point mutations II disrupted complex formation. Furthermore, immunofluorescence studies showed that these mutations abolished co-localization of Rab33B and Atg16L1 in cells. The Rab33B binding site of Atg16 identified in this study comprises residues 191-208 and is in close proximity of the PROPPIN WIPI2B binding site (207-230) and could explain how Golgi-derived vesicles can be recruited into close proximity of the isolation membrane by binding of Atg16 to both Rab33B and WIPI2B, providing a source of lipids to the growing isolation membrane. In my third project, I characterized the SCOC-FEZ1 complex that has a regulatory role in autophagy. Complex formation is mediated through the dimeric coiled coil domains of both proteins. Crystals diffracting up to 2.2 Å resolution were obtained but due a twinning problem the structure could not be determined. However, I gained new insights into SCOC-FEZ1 complex formation through biophysical experiments. I showed that the two dimers interact with a 1:1 stoichiometry with SEC-MALLS experiments. Cross-linking and analysis by mass spectroscopy revealed that FEZ1 most likely a forms parallel coiled coil dimer and that the SCOC and FEZ1 dimers interact in a parallel orientation with each other.2018-05-2