Structure and Function of Ubiquitin Conjugating Enzyme E2-25K:  The Tail Is a Core-Dependent Activity Element

Abstract

Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.

Proximity-induced activation of human Cdc34 through heterologous dimerization

“…Previous studies have shown that dimerization of the E2-25K core (amino acids 1-153) by GST fusion markedly enhanced the catalytic synthesis of polyUb chains by the truncated enzyme (18). However, this activation apparently proceeded through a mechanism involving formation of thiol-linked Ub chains that was not observed with GST-fused full-length E2-25K (18).…”

“…However, this activation apparently proceeded through a mechanism involving formation of thiol-linked Ub chains that was not observed with GST-fused full-length E2-25K (18). For this reason, it is unclear whether GST-Cdc34 and GST-E2-25K (1-153) use the same mechanism to mediate their increased ubiquitination activity.…”

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

“…The molecular functions of only a handful of these extensions are known [38][39][40][41] , and to date, the structural basis for protein-protein interactions mediated by these extensions remain elusive. One of the best understood extensions is in the E2 Ubc2p, which interacts with the E3, Ubr1p.…”

Solution Conformation of Lys63-linked Di-ubiquitin Chain Provides Clues to Functional Diversity of Polyubiquitin Signaling

“…Yeast Mms2 and Ubc13 were expressed and purified as in previous studies (21,22). Ub (K63C), Ub (K63R), and Ub (Asp 77 ) were expressed and purified as described in Haldeman et al (23). 15 N-Labeled proteins were expressed in Escherichia coli cells grown in minimal media with 15 NH 4 Cl as the sole source of nitrogen.…”