MUL1-RING recruits the substrate, p53-TAD as a complex with UBE2D2-UB conjugate

The RING domain of MUL1 (RING(MUL1)) alone mediates ubiquitylation of the p53-transactivation domain (TAD(p53)). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TAD(p53) by RING(MUL1), we determined the complex structure of RING(MUL1):UBE2D2 and studied the interaction between RING(MUL1) and TADp53 in the presence of UBE2D2-UB thioester (UBE2D2 similar to UB) mimetics. The RING(MUL1)-binding induced the closed conformation of UBE2D2(S22R/C85S)- UBK48R oxyester (UBE2D2(RS)- (UBOE)-O-R), and strongly accelerated its hydrolysis, which was suppressed by the additional N77Amutation of UBE2D2. Interestingly, UBE2D2(S22R/N77A/C85S)- (UBR)-R-K48 oxyester (UBE2D2(RAS)- (UBOE)-O-R) already formed a closed conformation in the absence of RING(MUL1). Although TAD(p53) exhibited weak binding for RING(MUL1) or UBE2D2 alone, its binding affinity was enhanced and even further for RING(MUL1):UBE2D2 and RING(MUL1):UBE2D2(RAS)-(UBOE)-O-R, respectively. The recognition of TAD(p53) by RING(MUL1) as a complex with UBE2D2 similar to UB is related to the multivalency of the binding events and underlies the ability of RING(MUL1) to ubiquitylate the intrinsically disordered protein, TAD(p53).