A Structurally Unique E2-Binding Domain Activates Ubiquitination by the ERAD E2, Ubc7p, through Multiple Mechanisms

Abstract

SUMMARY Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the ‘backside’ of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This is the first example where an essential component of E3 complexes both binds to E2 and enhances E2 loading with ubiquitin. These findings provide new insights into mechanisms of stimulating ubiquitination.

Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

“…1E). The model was based on a superimposition of the Ubc7 crystal structure [Protein Data Bank (PDB) ID code 4JQU] and a solution structure of the Doa10 RING domain (PDB ID code 2M6M) with the structure of the RING finger protein 4 (RNF4) RING:UbcH5B∼Ub complex (PDB ID code 4AP4) (3,19). The resulting in silico model predicted a hydrogen bond between the side chain of Lys118 and the backbone carbonyl of Leu8 of Ub (Fig.…”

“…Consequently, to ensure a single-round Ub transfer, we used preformed thiol esters of Ubc7:Cue1 with FlagUb R48 as donor Ub species, and Ubc7 (without Cue1), charged with wild-type Ub, as acceptor Ub species. Because FlagUb R48 can be conjugated to an acceptor Ub but then blocks further Lys48-linked conjugation, it serves only as a donor, whereas in the absence of Cue1, Ubc7 serves solely as an acceptor (19), because it cannot transfer the active-site bound Ub (23,26). Accordingly, purified Ubc7 or Ubc7 R118 in complex with Cue1 initially was charged with FlagUb R48 and, in parallel wild-type Ubc7 purified in the absence of Cue1, was charged with wild-type Ub (Fig.…”

“…The shared E2 enzyme, Ubc7, is a soluble cytosolic protein whose binding to either of the E3-ligase complexes at the ER membrane is mediated by the auxiliary ER membrane protein Cue1. Binding to Cue1 not only mediates the interaction with the E3-ligase complex but also protects Ubc7 from degradation and stimulates its Ub-transfer activity (16)(17)(18)(19)(20). Ubc7 is highly conserved in evolution, as evident from substantial sequence and structure similarities with its orthologs from other species (21).…”

“…Ubc7 and Ube2g2 are subjected to similar regulatory mechanisms: They bind to and are activated by the RING domains of their cognate E3s as well as by the E2-binding regions and CUE domains within Cue1 and gp78 (19,20,(23)(24)(25)(26). The evolutionarily conserved sequence, structure, and regulatory mechanisms of the Ubc7 E2s imply an essential physiological function.…”

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase

“…The activity of Ubc7 is largely governed by binding to its partner protein Cue1, probably to prevent uncontrolled ubiquitylation and substrate degradation Bazirgan and Hampton, 2008;Biederer et al, 1997;Kostova et al, 2009;Metzger et al, 2013;Ravid and Hochstrasser, 2007). Both the requirement for preceding Ubc6-mediated priming in the Doa10 ligase pathway and the unique E2 binding sites on the E3 RING finger domain constitute additional regulatory elements for the activity of…”