E1-E2 interactions in ubiquitin and Nedd8 ligation pathways.

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 ß-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 ß-grasp domains between ubiquitin and Nedd8 activating enzymes fails to reverse the effect of truncation. Thus, the conserved Helix 1 binding motif and the ß-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.

Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme.

Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP.Mg(2+) binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp(576) or Lys(528) results in dramatically impaired binding affinities for ATP.Mg(2+), a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k(cat) values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp(576) and Lys(528) are important for ATP.Mg(2+) binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp(576) within the activating enzymes for other ubiquitin-like proteins.

Protein interactions within the N-end rule ubiquitin ligation pathway.

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.