Study of the Lysine Deprotonation Mechanism in Ubiquitin Conjugating Enzyme Ubc13

Abstract

Ubiquitin (Ub) is a regulatory protein with the ability to flag proteins to be degraded by the proteosome. Ub is covalently attached to a lysine on the target protein by a series of reactions catalyzed by three types of enzymes: ubiquitin activating enzymes, E1; ubiquitin conjugating enzymes, E2; and ubiquitin ligases, E3. Before Ub is transferred to its target, it is bonded to the E2 via a thioester linkage. In this study, we examine the E2 enzyme, Ubc13, which catalyzes the formation of K63-linked polyubiquitin chains. The chains are formed when a lysine on the target Ub (K63) attacks the thioester bond between Ubc13 and the substrate Ub. To initiate this reaction, K63 on the target Ub must be deprotonated, turning it into an active nucleophile. There are two possible deprotonation sites: a conserved aspartate in Ubc13 (D119) and a conserved glutamate in the target ubiquitin (E64). In order to determine any preference between D119 and E64, we used classical molecular dynamics, Born-Oppenheimer molecular dynamics and single point QM/MM to model K64 deprotonation in several E2∼Ub conjugates.