Mechanism of E1-E2 Interaction for the Inhibition of Ubl Adenylation

Conjugates of ubiquitin or itshomologues to other proteins occur bystrictly ordered steps with orderedaddition of substrates for each step.High concentrations of E2 were shownto inhibit the formation of E2~Ublthioester and Ubl~target conjugates.We investigated the mechanism ofsuch inhibitory effect of the SUMO E2,and whether the E2 has two bindingsites on its E1, one for the inhibitoryeffect and one for the productiveSUMOylation. NMR methods incombination with mutagenesis andbiochemical assays revealed that Ubc9binds to two flexible domains of itsfree E1 simultaneously, suggestingextensive domain movements in thefree E1. Further, interaction of freeE1 and E2 inhibits SUMO adenylation,and the interfaces responsible for theinhibition were the same as thoserequired for productive transfer ofSUMO from E1 to E2. This studyindicates a conformational flexibilitydependent mechanism to control thestrictly ordered steps in Ublmodifications.