Homology building, protein-protein docking and MD approach to the simulation of the quaternary complex E1/E2/Ub2

Ubiquitylation is essential to numerous cellular and developmental processes. Alterations in this process are observed in most cancer cells. Ubiquitylation is catalysed by the sequential action of ubiquitin-activating enzyme (UbA1; E1), a ubiquitin-conjugating enzyme (UbC; E2) and a ubiquitin protein ligase (E3). Despite the central role of the E1 enzyme in the Ub conjugation cascade, and its potential as drug target, information regarding the mechanism of UbA1 ubiquitylation or of the Ub transfer from UbA1 to the E2, are limited to mutational studies,[1] and the mechanistic details of ubiquitin thioester bond formation and transfer to substrate are still undetermined. In this context, we have simulated in silico the entire process leading to the UbA1-UbC interaction, by means of a computational approach including many techniques such as homology building, protein-protein docking and molecular dynamics. In particular here we describe static models of hUbA1, as well as of its intermediate complexes involved in the activation and in the formation of transient thiolester complex: i) hUbA1 in complex with adenylated ubiquitin, ii) the covalent adduct between Ub and hUbA1, iii) the hUbA1 in complex with adenylated ubiquitin and covalently linked to the ubiquitin and, finally iv) the quaternary complex including hUbA1 two ubiquitin molecules and a member of the UBC family of enzymes. In this proposal, we intend to study the conformational behaviour of these four model by using a computational approach characterized by an increased level of accuracy in the study of the system. To the best of our knowledge, this is the first structural information on the hUbA1/E2 complex, and therefore could disclose important information on the mechanism of this important enzyme and will pave the way to the rational design of ubiquitylation inhibitors.