Fig. 6. Model of E1–E2 thioester transfer reaction cycle.

a Ub E1 catalyzes adenylation of the Ub C-terminal Gly76 in the presence of ATP and Mg²⁺ b Thiolation with E1 catalytic cysteine through disassemble of adenylation domain and a ~130° rotation of SCCH domain. c E1–Ub thioesterification achieved by SCCH relocation to the original open position. d A second Ub binds in the adenylation active site of E1–Ub to form doubly loaded E1–Ub(t)/Ub(a). e A E2 is recruited to the UFD with distal configuration in doubly loaded E1–Ub(t)/Ub(a). f UFD rotates from distal to proximal to bring E1 E2 catalytic cysteines into close proximity for E2–Ub thioester bond formation. g Ub(t) is transferred from E1 catalytic cysteine to E2 catalytic cysteine. h E2–Ub thioester product is released from E1 with UFD domain in E1 rotating back to the distal conformation to continue the reaction cycle.