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.
Bibliographical noteFunding Information:
We thank J. Hurley (National Institute of Diabetes and Digestive and Kidney Diseases) for the Ubc13–Mms2 coexpression plasmid and X. Zhang (Johns Hopkins University) for human E1 protein. We also thank J. Stivers, A. Hengge and L. Spyracopoulos for helpful discussions. This work was supported in part by a grant from the US National Science Foundation (MCB-0920082). C.E.B. was supported in part by a Ruth Kirchstein Fellowship from the National Institute of General Medical Science (F32GM089037). General Medical Sciences and Cancer Institutes Structural Biology Facility at the Advanced Photon Source has been funded in whole or in part with Federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357.