S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity

Abstract

Summary Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1 interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N-termini are serine/threonine-rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1

“…Comparison of the Uba1 SCCH_ALT structure with previous Uba1 structures with the SCCH domain in the open conformation (hereafter referred to as Uba1 SCCH_OPEN ) (14,19,20,22) reveals that the SCCH domain has undergone a domain alternation defined by a 106°rigid body rotation that translocates the catalytic cysteine residue (Cys 593 ) 34 Å (Fig. 1A).…”

“…The Cys domain, which is split into first and second catalytic cysteine half-domains (FCCH and SCCH domains, respectively), harbors the catalytic cysteine residue that forms the thioester bond with Ub/Ubl (14 -17). Finally, the ubiquitinfold domain (UFD) of a canonical E1 is involved in molecular recognition of its cognate E2(s), which is followed by E1-E2 thioester transfer and formation of the E2ϳUb thioester intermediate (17)(18)(19)(20).…”

“…To date, five crystal structures of Uba1 have been reported in which all nine copies of Uba1 are observed with the SCCH domain in the open conformation with the catalytic cysteine 34 -37 Å away from the C terminus of Ub (14,19,20,22). Although previous studies suggest that the structural basis for Figure 2.…”

“…WT and mutated variants of full-length (residues 1-1012) S. pombe Uba1, S. pombe Ubc4, and S. pombe Ub (residues 1-76) used in structural and biochemical studies were expressed in Escherichia coli and purified as described previously (19). Purified proteins were concentrated to ϳ25 (Uba1 WT and variants) or ϳ10 mg/ml (Ub) and snap frozen in liquid nitrogen for use in subsequent crystallization and biochemical experiments.…”

New ubiquitin-dependent mechanisms regulating the Aurora B–protein phosphatase 1 balance in Saccharomyces cerevisiae

“…Given this hierarchy, interest in specific ubiquitindependent processes has logically focused on E2 and E3 enzymes. However, the structures of several E1-E2 complexes indicate that different E2s associate with unique structural elements of the E1 (Olsen and Lima, 2013;Lv et al, 2017), suggesting a potential regulatory role for the E1 enzyme. In addition, Uba1, the sole E1 enzyme in Saccharomyces cerevisiae, is post-translationally modified by phosphorylation, ubiquitylation and succinylation at multiple sites (Albuquerque et al, 2008;Holt et al, 2009;Swaney et al, 2013;Fang et al, 2014), providing potential mechanisms for regulating specific interactions with E2 enzymes.…”