title: Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate.
abstract: During ubiquitin conjugation, the thioester bond that links "donor" ubiquitin to ubiquitin-conjugating enzyme (E2) undergoes nucleophilic attack by the ɛ-amino group of an acceptor lysine, resulting in formation of an isopeptide bond. Models of ubiquitination have envisioned the donor ubiquitin to be a passive participant in this process. However, we show here that the I44A mutation in ubiquitin profoundly inhibits its ability to serve as a donor for ubiquitin chain initiation or elongation, but can be rescued by computationally predicted compensatory mutations in the E2 Cdc34. The donor defect of ubiquitin-I44A can be partially suppressed either by using a low pKa amine (hydroxylamine) as the acceptor or by performing reactions at higher pH, suggesting that the discharge defect arises in part due to inefficient deprotonation of the acceptor lysine. We propose that interaction between Cdc34 and the donor ubiquitin organizes the active site to promote efficient ubiquitination of substrate.
authors.affiliation: Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Anjanabha
authors.initials: A
authors.lastname: Saha
authors.forename: Steven
authors.initials: S
authors.lastname: Lewis
authors.forename: Gary
authors.initials: G
authors.lastname: Kleiger
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D020134:Catalytic Domain; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C577447:CDC34 protein, human; C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome