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.
Highlights
► Determined crystal and NMR structures of E4B U box demonstrating a monomeric state ► Demonstration of a hydrogen-bond network in E4B U box via NMR scalar couplings ► First 3D structure of a monomeric U box–E2 enzyme complex (E4B U box bound to UbcH5c) ► Evidence for an allosteric effect of E4B U box on UbcH5c and Ubc4 E2 enzymes
