title: E1-E2 interactions in ubiquitin and Nedd8 ligation pathways.
abstract: Initial rates of E1-catalyzed E2 transthiolation have been used as a reporter function to probe the mechanism of 125I-ubiquitin transfer between activation and ligation half-reactions of ubiquitin conjugation. A functional survey of 11 representative human E2 paralogs reveals similar Km for binding to human Uba1 ternary complex (Km(ave)=121±72 nm) and kcat for ubiquitin transfer (kcat(ave)=4.0±1.2 s(-1)), suggesting that they possess a conserved binding site and transition state geometry and that they compete for charging through differences in intracellular concentration. Sequence analysis and mutagenesis localize this binding motif to three basic residues within Helix 1 of the E2 core domain, confirmed by transthiolation kinetics. Partial conservation of the motif among E2 paralogs not recognized by Uba1 suggests that another factor(s) account for the absolute specificity of cognate E2 binding. Truncation of the Uba1 carboxyl-terminal β-grasp domain reduces cognate Ubc2b binding by 31-fold and kcat by 3.5×10(4)-fold, indicating contributions to E2 binding and transition state stabilization. Truncation of the paralogous domain from the Nedd8 activating enzyme has negligible effect on cognate Ubc12 transthiolation but abrogates E2 specificity toward non-cognate carrier proteins. Exchange of the β-grasp domains between ubiquitin and Nedd8 activating enzymes fails to reverse the effect of truncation. Thus, the conserved Helix 1 binding motif and the β-grasp domain direct general E2 binding, whereas the latter additionally serves as a specificity filter to exclude charging of non-cognate E2 paralogs in order to maintain the fidelity of downstream signaling.
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors: Zeynep
authors: Z
authors: Tokgöz
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors: Thomas J
authors: TJ
authors: Siepmann
authors: Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112.
authors: Frederick
authors: F
authors: Streich
authors: Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112.
authors: Brajesh
authors: B
authors: Kumar
authors: Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112.
authors: Jennifer M
authors: JM
authors: Klein
authors: Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112; Stanley S. Scott Cancer Center, Louisiana State University School of Medicine, New Orleans, Louisiana 70112. Electronic address: ahaas@lsuhsc.edu.
authors: Arthur L
authors: AL
authors: Haas
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000075687:NEDD8 Protein; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D025801:Ubiquitin; D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes