Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin

Abstract

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1β1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

“…Through structural and mutagenesis studies, we identify a novel and conserved mechanism through which canonical PCGF2/4 limits the activity of RING1. In addition, we demonstrate that, contrary to expectation 6,30 , PCGF-RING1 ligases use a mode of Ub recognition and activation 31 highly similar to that recently discovered for homodimeric RING E3s 30,32,33 and monomeric RINGs CBL-B and RNF38 34,35 . In contrast to the differences in their intrinsic catalytic activities, we find all the six PCGF-RING1 heterodimers to be robust H2A ligases in an assay using purified components.…”

“…Structures of RING E3s in complex with charged UbcH5-family E2s reveal a consistent arrangement of the E2, the RING and Ub 30,31,[33][34][35] ( Fig. 3; Supplementary Fig.…”

“…We asked whether the PCGF high-activity group complexes might exhibit enhanced affinity for E2BUb. Because the E2BUb thiolester bond is highly labile, we generated a stable isopeptide mimic of Ub-charged E2 from UbcH5c carrying the mutations C85K (to permit isopeptide bond formation) and S22R (to block an unwanted secondary interaction between Ub and UbcH5c 35,44 ). The dissociation constant for E2 or E2BUb binding to each immobilized E3 complex was determined using bio-layer interferometry (BLI; Table 3, Supplementary Fig.…”

“…3f,g) suggest features reminiscent of both the homodimeric RING and p-CBL-B structures. Like RNF4 and BIRC7, the Ub contact surface includes residues from the E2-distal RING, in this case from the PCGF subunit helix a3, and like CBL-B, the primary contact is charged rather than aromatic (see also the recent RNF38 example 35 ). For both PCGFs, there is a negatively charged residue roughly in the position of the phosphotyrosine of p-CBL-B (see E77 in Fig.…”

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

“…Other than at the E2 active site, ubiquitin also binds to a specific region on the "backside" of a subset of E2s (Ub B binding), which enhances RING:E2 affinity and stimulates ubiquitination (17)(18)(19)(20). Several E3s also bind their cognate E2s on secondary sites away from the RING-E2 interface.…”