The lysine deprotonation mechanism in a ubiquitin conjugating enzyme

Ubiquitination is a biochemical reaction in which a small protein, ubiquitin (Ub), is covalently linked to a lysine on a target protein. This type of post-translational modification can signal for protein degradation, DNA repair, or inflammation response. Ubiquitination is catalyzed by three families of enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). In this study, we focus on the chemical mechanism used by the E2 enzyme, Ubc13, which forms polyubiquitin chains by linking a substrate Ub to Lys63 on a target ubiquitin (Ub*). Initially, Ubc13 is covalently linked to the substrate Ub. Next, Lys63 in the Ub* is deprotonated, becomes an active nucleophile, and attacks the thioester bond in the Ubc13~Ub conjugate. The deprotonation mechanism is not well understood. There are two, conserved nearby residues that may act as conjugate bases (Asp119 on Ubc13 and Glu64 on Ub*.) It is also hypothesized that the active site environment suppresses the lysine's pKa, favoring deprotonated lysine. We test these hypotheses by simulating both WT and mutant Ubc13 with constant pH molecular dynamics (CpHMD), which allows titratable residues to change their protonation states. We use these simulations to monitor the protonation states and to generate titration curves of lysine 63. We found that the pKa of the lysine is highly dependent on its distance from the active site. Also, mutating Asp119 or Glu64 to Ala has little effect on the lysine pKa, indicating that neither residue acts as a generalized base. Finally, we note that mutating a structural residue (Asn79 to Ala) increases the lysine pKa, suggesting that alterations to the active site hydrogen bonding network can affect nucleophile activation.