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

Abstract

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 TAD_p53 in the presence of UBE2D2-UB thioester (UBE2D2~UB) mimetics. The RING_MUL1 -binding induced the closed conformation of UBE2D2^S22R/C85S -UB^K48R oxyester (UBE2D2^RS -UB^R _OE ), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A-mutation of UBE2D2. Interestingly, UBE2D2^{S22R/N77A/C85S} -UB^K48R oxyester (UBE2D2^RAS -UB^R _OE ) 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 -UB^R _OE , respectively. The recognition of TAD_p53 by RING_MUL1 as a complex with UBE2D2~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 .

Keywords: MUL1 RING domain; NMR; UBE2D2; p53 transactivation domain; ubiquitin.