An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair.
The human CCR4-NOT complex is a global regulator of RNA polymerase II transcription. Recently, we showed that the RING domain CNOT4 subunit contains intrinsic ubiquitin-protein ligase (E3) activity. Here we show that binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective. To understand the basis for this interaction, we identified several basic residues of UbcH5B important for binding to CNOT4 by mutational analysis. Subsequently, we tested pairs of UbcH5B and CNOT4 mutants for restoration of interaction. Concomitant charge-alteration of E49 of CNOT4 and K63 of UbcH5B restored binding and re-created a functional enzyme pair, indicative of an electrostatic interaction between these residues. The corresponding amino acids in the yeast orthologues can also be used to create a similarly designed E2-E3 enzyme pair. These are the first examples of altered-specificity E2-E3 enzyme pairs and give further insight into how E2-E3 specificity is obtained.
Department of Physiological Chemistry, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
G Sebastiaan
GS
Winkler
Thomas K
TK
Albert
Cyril
C
Dominguez
Yvonne I A
YI
Legtenberg
Rolf
R
Boelens
H Th Marc
HT
Timmers
D019943:Amino Acid Substitution; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D046911:Macromolecular Substances; D009154:Mutation; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C539882:NR4A2 protein, human; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D014157:Transcription Factors; C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes