Differential ubiquitin binding by the acidic loops of Ube2g1 and Ube2r1 distinguishes their K48-ubiquitylation activities [Protein Synthesis and Degradation]

December 3rd, 2014 by Choi, Y.-S., Lee, Y.-J., Lee, S.-Y., Shi, L., Ha, J.-H., Cheong, H.-K., Cheong, C., Cohen, R. E., Ryu, K.-S.

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize K48-linked poly-ubiquitins without an E3 ligase, but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184-196) for efficient K48-ubiquitylation activity. The presence of Y102 and Y104 in the Ube2g1 acidic loop enhanced both ubiquitin-binding and K48-ubiquitylation, and distinguished Ube2g1 from the otherwise similar truncated Ube2r11-183 (Ube2r1C). Replacement of Q105-S106-G107 in the acidic loop of Ube2r1C (Ube2r1CYGY) by the corresponding residues from Ube2g1 (Y102-G103-Y104) increased K48-ubiquitylation activity and ubiquitin binding. Two E2~UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin-binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2~UB thioester. The ubiquitin moiety of the Ube2r1CC93S-15NUBK48R oxyester displayed two-state conformational exchange whereas the Ube2r1CC93S/YGY-15NUBK48R oxyester showed predominantly one state. Together with NMR studies that compared UBK48R oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intra-molecular interaction between the acidic loop and the attached donor ubiquitin regulates K48-ubiquitylation activity.
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