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Updated: Aug 30, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Dimerization of the human E3 ligase CHIP via a coiled-coil domain is essential for its activity
Rainer Nikolay1, Thomas Wiederkehr, Wolfgang Rist
1Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Abstract:
The Hsp70-interacting E3-ubiquitin ligase CHIP has been implicated in the decision as to whether a target protein enters the refolding or the degradation pathway. To further characterize the activity of CHIP we purified untagged Homo sapiens and Drosophila melanogaster CHIP (hCHIP, dCHIP). In contrast to other E3-ubiquitin ligases, both hCHIP and dCHIP proteins formed homodimers at physiological concentrations. We identified a predicted coiled-coil region in a mixed charge segment of the hCHIP and dCHIP sequence and found it to be necessary and sufficient for dimer formation. A mutant of hCHIP lacking this segment (hCHIPdelta-(128-229)) was incapable of dimer formation, but the segment by itself (hCHIP-(128-229)) readily dimerized. Furthermore, we demonstrated that dimerization is a prerequisite for activity of hCHIP in the reconstituted ubiquitination assay. Control of dimerization may thus provide a mechanism for regulation of CHIP activity.
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