Functional insight and Mechanistic Study of UBA domain of E2-25K in Lys-48 Ubiquitin Chain Elongation

Gajendra Singh

E2-25K (Ube2K) is a ubiquitin-conjugating enzyme that can only synthesize the K48(Lys-48) ubiquitin chain. All ubiquitin E2s have a conserved catalytic ubiquitin-conjugating domain (UBC), whereas E2-25K is the only enzyme that contains additional C-terminal ubiquitin association domain (UBA). We investigated the function of UBA domain in K48 chain elongation. NMR based results suggest that UBA domain provides the binding surface to ubiquitin during the chain elongation. It also holds the di ubiquitin (Ub 2) with different affinity to proximal and distal Ub units. We showed that the UBA domain expedites the polyubiquitin (K48) chain formation. Fluorescent polarization and mutational studies indicated that the UBA domain increases the K48 chain processivity. However, the activity is dependent upon the Ub chain length. Gel-based kinetics results also manifest the function UBA domain in polyubiquitin chain and the results evident that the rate reduces, drastically with truncated UBA domain.