Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

다국어 초록 (Multilingual Abstract)

<P>The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins 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 Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1<SUP>1–183</SUP> (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1C<SUP>YGY</SUP>) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-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 Ube2r1C<SUP>C93S</SUP>-[<SUP>15</SUP>N]UB<SUP>K48R</SUP> oxyester displayed two-state conformational exchange, whereas the Ube2r1C<SUP>C93S/YGY</SUP>-[<SUP>15</SUP>N]UB<SUP>K48R</SUP> oxyester showed predominantly one state. Together with NMR studies that compared UB<SUP>K48R</SUP> oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, <I>in vitro</I> ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.</P>