SUMMARY The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an ~ 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and consequently ubiquitylation.

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING: Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2: G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination.

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.