Crystal Structure of UbcH5b∼Ubiquitin Intermediate: Insight into the Formation of the Self-Assembled E2∼Ub Conjugates

Abstract

E2 ubiquitin-conjugating enzymes catalyze the attachment of ubiquitin to lysine residues of target proteins. The UbcH5b E2 enzyme has been shown to play a key role in the initiation of the ubiquitination of substrate proteins upon action of several E3 ligases. Here we have determined the 2.2 A crystal structure of an intermediate of UbcH5b~ubiquitin (Ub) conjugate, which is assembled into an infinite spiral through the backside interaction. This active complex may provide multiple E2 active sites, enabling efficient ubiquitination of substrates. Indeed, biochemical assays support a model in which the self-assembled UbcH5b~Ub can serve as a bridge for the gap between the lysine residue of the substrate and the catalytic cysteine of E2.

Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

“…Previously, two residues of E2 were shown to be important in E2 catalyzed isopeptide bond synthesis, but not in HECT ligase catalyzed Ub conjugation (21,(27)(28)(29). The "catalytic asparagine" (N77 in UbcH5a) is important in stabilizing the oxyanion intermediate during RING E3 mediated conjugation (27); and D117 in UbcH5b (or D127 in UbcH9) facilitates efficient Ub and SUMO transfer from E2 to lysine residues of substrates (15,17,21,28). In the UbcH7/NleL structure, the corresponding residues (N78 and H119) are located within 10 Å of the E3 catalytic cysteine C753 (Fig.…”

Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

“…Specifically, solutionbased two-dimensional NMR studies detected Ub bound noncovalently near Ser 22 on human (Homo sapiens (Hs)) UbcH5c (12), which has been confirmed by activity assays (13,14) and crystallography (14) for HsUbcH5b. Noncovalent Ub-E2 interaction is proposed to aid assembly of Ub-E2 polymeric complexes for enhanced target protein polyubiquitylation (12,14). Ub enzyme variants (UEVs) resemble E2s but lack an active site cysteine, thus precluding covalent conjugation to Ub-like proteins.…”

“…HsUbcH5c noncovalent interaction with Ub and the subsequent formation of high molecular weight (UbϳE2) n polymers are required for auto-polyubiquitylation of the E3 BRCA1 (12). Here, the S22R mutant of HsUbcH5c is unable to support (UbϳE2) n polymer formation but can still sustain auto-monoubiquitylation of BRCA1 in vitro (12)(13)(14)45). The shared use of the E2 noncovalent interaction surface by MUB and Ub poses several possibilities where MUBs influence Ub chain formation (Fig.…”

“…evaluations of Mms2 and HsUbcH5 with Ub were performed with sensitive NMR-based methods (12,14,16). However, this does raise the possibility that MUBs bind Subgroup VI E2s with greater affinity than the Ub-E2 interactions described previously.…”

Structural Determinants of Ubiquitin Conjugation in Entamoeba histolytica

“…The resulting E1ϳUb thioester complex engages a ULM-conjugating enzyme (E2), with major contributions to the E1/E2 interface arising from the E1 ubiquitin-fold domain (UFD) (5). The thioester bond between the ubiquitin C terminus and the E1 enzyme is transferred to a conserved cysteine residue on the E2, resulting in a covalent E2ϳUb adduct (6). E3 ubiquitin-ligating enzymes can be classified into two major groups, containing either homology to the E6AP C terminus (HECT) or really interesting new gene related (RING) domains (3).…”