E6AP/UBE3A Ubiquitin Ligase Harbors Two E2∼ubiquitin Binding Sites

Abstract

Background:The mechanism of E6AP has been previously defined by the structure of a substrate-bound intermediate. Results:Rate studies of polyubiquitin chain formation preclude the canonical model based on the crystal structure. Conclusion: Kinetics define a mechanism requiring two functionally distinct E2ϳubiquitin thioester binding sites. Significance: A general mechanism for Hect ligase polyubiquitin chain formation is defined for E6AP based on empirical rate measurements.

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

“…The signal that accumulates in the resolving gel in the presence of UbcH6, UbE2E2, UbcM2, or E2 epf (Fig. 1A, lanes 6 -8 and 10) results from IpaH9.8-independent autoconjugation of the E2 paralogs (32,39). These results confirm that members of the Ubc5 family of E2 paralogs are the cognate carrier proteins supporting IpaH9.8-catalyzed 125 I-polyubiquitin chain formation.…”

“…Intensity values for the GST-tagged enzymes were corrected for the additional binding of dye due to the GST moiety under the assumption that GST and IpaH9.8 exhibit similar color yields for Coomassie dye binding. 48 -linked Polyubiquitin Chains-All E3 ubiquitin ligases tested to date are capable of assembling polyubiquitin chains in the absence of their cognate target proteins (32,39,42). The initial rates of polyubiquitin chain formation can thus be exploited as a facile reporter function for detailed kinetic analysis of the E3 ligase mechanism even without knowledge of the cognate target protein(s) or post-translational modification(s) required for target protein recognition (32,39,42).…”

“…48 -linked Polyubiquitin Chains-All E3 ubiquitin ligases tested to date are capable of assembling polyubiquitin chains in the absence of their cognate target proteins (32,39,42). The initial rates of polyubiquitin chain formation can thus be exploited as a facile reporter function for detailed kinetic analysis of the E3 ligase mechanism even without knowledge of the cognate target protein(s) or post-translational modification(s) required for target protein recognition (32,39,42). Previous reports suggest that IpaH9.8 functions solely with the Ubc5 family of E2-conjugating enzymes (17,20,22); however, differences in the stability of various E2 families, ambiguity associated with establishing the rate-limiting step in such screens, and errors in estimating active E2 concentration based on total protein complicate such an assignment, as discussed previously (42).…”

“…It also allows comparison of the IpaH mechanism with features of eukaryotic HECT domain ligases potentially to discover functional aspects of the enzymes by analogy. Recent findings from our group have revealed previously unsuspected complexity in the mechanism of the HECT ligase-catalyzed polyubiquitin chain formation (32,33). Kinetic studies show that E6AP possesses two E2ϳubiquitin thioester binding sites that function in concert to assemble the polyubiquitin degradation signal, which presumably remains tethered to the active site cysteine of the HECT domain as a thioester until en bloc transfer to the targeted substrate (32,34).…”

“…Recent findings from our group have revealed previously unsuspected complexity in the mechanism of the HECT ligase-catalyzed polyubiquitin chain formation (32,33). Kinetic studies show that E6AP possesses two E2ϳubiquitin thioester binding sites that function in concert to assemble the polyubiquitin degradation signal, which presumably remains tethered to the active site cysteine of the HECT domain as a thioester until en bloc transfer to the targeted substrate (32,34). More recent kinetic evidence identifies a radial symmetric E6AP trimer as the catalytically competent form of the enzyme (33).…”