A conserved asparagine has a structural role in ubiquitin-conjugating enzymes

Abstract

It is widely accepted that ubiquitin-conjugating enzymes contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13-Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.

Figure 1. Polyubiquitin chain formation by Ubc13/Mms2 in the presence and absence of Rad5

(a) Substrate partitioning experiments showing active fraction of Ubc13~Ub thioester as a function of acceptor ubiquitin concentration. Plot of percentage diubiquitin formed versus concentration of ubiquitin (Ub)^{Δ 75,Δ76} for Ubc13^N79Q-Mms2 (filled circles) or Ubc13^N79Q-Mms2 with the Rad5 RING domain (open circles). Points represent the average of 3 to 4 separate measurements with the standard deviation shown by the error bars. (b) Single discharge assays of diubiquitin formation by Ubc13-Mms2 containing wild-type Ubc13 or mutants with substitutions at N79, performed in the presence and absence of the Rad5 RING fragment. Full gels in Supplementary Figure 4.

Figure 2. Crystal structure of Ubc13^N79A

(a) Alignment of the structure of Ubc13^N79A (salmon) with wild-type Ubc13 from 2GMI (yellow). Residue names are colored to match the coloring of the N79A or wild-type structures. (b) Electron density 2F₀-F_c map contoured at 1.0σ showing the density for the active site loop in Ubc13^N79A. The backbone Cα trace of wild-type Ubc13 from 2GMI is shown in green. (c) Single discharge assay for the Ubc13 active site loop mutants using ubiquitin^Δ75,Δ76 (Ub) as the ubiquitin acceptor.