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J Biol Chem. 2017 Jun 23;292(25):10398-10413. doi: 10.1074/jbc.M117.789479. Epub 2017 May 1.

Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase.

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From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037.
the Departments of Cell and Molecular Biology and.
the Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, and.
From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037,


Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.


E3 ubiquitin ligase; HECT; polyubiquitin chain; protein degradation; ubiquitin; ubiquitylation (ubiquitination)

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