Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4.
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.
Benirschke
Robert C
RC
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Thompson
James R
JR
Nominé
Yves
Y
Wasielewski
Emeric
E
Juranić
Nenad
N
Macura
Slobodan
S
Hatakeyama
Shigetsugu
S
Nakayama
Keiichi I
KI
Botuyan
Maria Victoria
MV
Mer
Georges
G
D000494:Allosteric Regulation / Q000235:genetics; D002151:Calorimetry; D018360:Crystallography, X-Ray; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D025521:Tumor Suppressor Proteins; C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C473697:UBE4B protein, human; D044767:Ubiquitin-Protein Ligases