The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Abstract

Background: E6AP/UBE3A is a Hect ligase implicated in neurodevelopment and cell transformation. Results: Kinetic/biophysical analyses demonstrate that E6AP oligomerization is required for polyubiquitin degradation signal assembly and that changes in oligomerization regulates such activity. Conclusion: E6AP oligomerization accounts for opposing effects of mutation and HPV16/18 E6 protein.Significance: These findings explain in part the etiology of specific Angelman syndrome mutations and E6-mediated cell transformation.

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

“…The empirically derived Hill coefficients (n H ) for E2ϳubiquitin binding approach 2, indicating that IpaH9.8 functions minimally as a dimer. The only other published example of cooperativity among ubiquitin ligases is the TRIM superfamily of RING ligases (39), although non-cooperative oligomerization is also required for E6AP-catalyzed assembly of Lys 48 -linked polyubiquitin degradation signals (33). Cooperative substrate binding requires IpaH9.8 to function as an obligate oligomer.…”

“…Both have affinities for their cognate E2ϳubiquitin thioester co-substrates in the low nanomolar range (Table 2) (32) and k cat for polyubiquitin chain formation of 5-10 s Ϫ1 when corrected for negative cooperativity ( Table 2) or degree of oligomerization (33). Both ligase families additionally exhibit empirically validated mechanisms requiring ordered binding of the E2ϳubiquitin thioester co-substrate to two functionally distinct sites, the requirement of oligomerization for polyubiquitin chain formation, and previously documented requirements for ubiquitin thioester formation to conserved cysteine catalytic residues (22,24).…”

“…Each enzyme was present at 75 M total protein concentration in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT (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).…”

“…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).…”