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April 17th, 2025
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Tsinghua University
biochemistry
biorxiv

Structural Visualization of HECT-type E3 ligase Ufd4 Accepting and Transferring Ubiquitin to Form K29/K48-branched Polyubiquitination

Wu, X.Open in Google Scholar•Ai, H.Open in Google Scholar•Mao, J.Open in Google Scholar•Cai, H.Open in Google Scholar•Liang, L.Open in Google Scholar•Tong, Z.Open in Google Scholar•Zheng, Q.Open in Google Scholar•LIU, L.Open in Google Scholar•Pan, M.Open in Google Scholar

The K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal Ub to the active cysteine of Ufd4 for K29-linked branched ubiquitination. These structures not only provide mechanistic insights into the architecture of the Ufd4 complex but also provide structural visualization of branched ubiquitin chain formation by a HECT-type E3 ligase.

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