Elucidating specificity and mechanism of the ubiquitination machinery

• Applicant: Dr. Sonja Gisela Lorenz
• Subject Area: Biochemistry
• Term: from 2013 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 240707550

My research group aims to decipher how ubiquitin − a single, small protein − achieves specificity in regulating all aspects of eukaryotic cell biology. The main thrust of my research program is the structural and mechanistic analysis of HECT-type ubiquitin ligases and the dynamic macromolecular complexes in which they engage. Over the past four years, my group has made significant progress in understanding the structural mechanisms of HECT ligases by interrogating their conformational dynamics, regulation, as well as substrate and ubiquitin recognition. For instance, we reported the first structural mechanism of regulation for the tumor-associated ligase HUWE1 (eLife, 2017) and determined the structural basis of ubiquitin recognition by E6AP with important implications for how individual HECT ligases generate diversity in ubiquitin linkage formation (in review). We have also succeeded in establishing a novel technique that enables us to elucidate the underpinnings of linkage specificity in HECT ligases at the atomic level – a major unresolved question in the field. Moreover, we have identified a regulation mechanism in UBE2S, a cognate ubiquitin-conjugating (E2) enzyme of the human the anaphase-promoting complex (APC/C). We are now requesting a final, one-year extension of funding by the Emmy Noether Programme in order to accomplish the following specific aims: (I) To elucidate the structural mechanism of substrate recognition by HUWE1, based on crystal structures of HUWE1-MIZ1 complexes we have determined(II) To determine the structural basis of substrate presentation to the catalytic domain and its impact on the conformational equilibrium of HUWE1 (III) To structurally delineate ubiquitin recognition by the catalytic domain of HUWE1, based on a crystal structure we have determined(IV) To elucidate the structural basis of linkage specificity in HUWE1 by determining the structure of a ternary complex containing HUWE1 and two ubiquitin molecules, building on the novel preparation strategy we have developed All of these projects are grounded in extensive preliminary data, with several crystal structures being already available. It is now crucial that we validate our structural and biochemical findings in cell-based analyses for publication in high-impact journals. To this end, a one-year extension of funding by the Emmy Noether Programme would be highly valuable.

• DFG Programme: Independent Junior Research Groups