From Beamline X29A: Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer.

Ubiquitin (Ub) conjugation is initiated by an E1 enzyme that catalyzes carboxy-terminal Ub adenylation, thioester bond formation to a catalytic cysteine in the E1 Cys domain, and thioester transfer to a catalytic cysteine in E2 conjugating enzymes. How the E1 and E2 active sites come together during thioester transfer and how Ub E1 interacts with diverse Ub E2s remains unclear. Here we present a crystal structure of a Ub E1-E2(Ubc4)/Ub/ATP.Mg complex that was stabilized by induction of a disulfide bond between the E1 and E2 active sites. The structure reveals combinatorial recognition of the E2 by the E1 ubiquitin-fold domain (UFD) and Cys domain and mutational analysis, coupled with thioester transfer assays with E1, Ubc4, and other Ub E2s, show that both interfaces are important for thioester transfer. Comparison to a Ub E1/Ub/ATP.Mg structure reveals conformational changes in the E1 that bring the E1 and E2 active sites together.

Figure: E1/Ub/ATP.Mg and E1-E2/Ub/ATP.Mg Structures(A) Cartoon of the Uba1/Ub/ATP.Mg complex with Uba1 domains color coded and labeled. E1 catalytic cysteine and ATP are shown as CPK-colored spheres and magnesium ions as cyan spheres. AAD and IAD stand for active and inactive adenylation domains, respectively; FCCH is first catalytic cysteine half domain.(B) Chemical structures of Ub/Ubl during E1-E2 thioester transfer (left) and the E1-E2 disulfide-linked complex (right). Active-site cysteine residues, E1, E2, and Ub/Ubl are labeled.(C) Three views of a cartoon representation of the Uba1-Ubc4/Ub/ATP.Mg complex labeled and colored as in (A). The disulfide bridge linking E1 and E2 cysteines is indicated by an arrow. A front view of the structure shown in the left panel, a top/down view in the center panel, and a side view in the right panel.

Results from:
Olsen SK, Lima CD.
Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer.
Mol Cell. 2013 Mar 7;49(5):884-96. doi: 10.1016/j.molcel.2013.01.013. Epub 2013 Feb 14.
PMCID: PMC3625138