Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.
Bibliographical noteFunding Information:
We acknowledge the use of beamline 19-BM at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source (APS). Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. We thank Younchang Kim at APS for assistance with X-ray data collection. We thank Ema Stokasimov for her early contribution to this project. Partial support from National Institutes of Health grant CA109449 and a March of Dimes Basil O'Connor scholarship to G.M. is gratefully acknowledged. J.R.T. acknowledges support from the Minnesota Partnership for Biotechnology and Medical Genomics. Y.N. was a recipient of a Kendall-Mayo postdoctoral fellowship in biochemistry.