E1-E2 Interactions in Ubiquitin and Nedd8 Ligation Pathways

Abstract

Background: Ubiquitin carrier protein (E2) recognition by ubiquitin activating enzyme (E1) defines fidelity in subsequent conjugation reactions. Results: E2 transthiolation kinetics identify structural features defining the specificity of E1-E2 binding. Conclusion: E2 paralogs contain a conserved E1 binding motif, and the E1 ␤-grasp domain is a specificity filter for E2 binding. Significance: This defines structural features determining ubiquitin conjugation fidelity.

Tripartite Motif Ligases Catalyze Polyubiquitin Chain Formation through a Cooperative Allosteric Mechanism

“…Active Uba1 was quantitated by the stoichiometric formation of 125 I-ubiquitin thioester (27,28). Recombinant human E2 proteins were those used previously (29), the activities of which were quantitated by the stoichiometric formation of E2-125 I-ubiquitin thioester and compared with the expected activities based on protein content determined by their calculated 280 nm extinction coefficients (10,28). Point mutants of Ubc5A were created using the QuikChange kit (Stratagene) (29,30).…”

“…Recombinant human E2 proteins were those used previously (29), the activities of which were quantitated by the stoichiometric formation of E2-125 I-ubiquitin thioester and compared with the expected activities based on protein content determined by their calculated 280 nm extinction coefficients (10,28). Point mutants of Ubc5A were created using the QuikChange kit (Stratagene) (29,30). Recombinant human IsoT (USP5) was expressed in Escherichia coli BL21(DE3) cultures harboring pRSIsoT (generous gift of Dr. Keith Wilkinson, Emory University School of Medicine) and purified to apparent homogeneity as described previously (31).…”

“…However, the construct was used to generate the GST-RING fusion by mutating the Leu 98 codon to a STOP codon to yield pGEX-TRIM32⌬CL98. The pGEX-TRIM32⌬CL98 plasmid was used to express the GST-RING fusion protein as described for GST-E2 proteins and subsequent thrombin cleavage of GST-RING yielded the RING domain alone (29). The GST moiety was indispensable for RING domain purification and quantitation because the RING domain has neither tryptophan nor tyrosine residues and, thus, no appreciable absorbance at 280 nm.…”

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

“…After 15 h, the cells were harvested by centrifugation at 6,000 ϫ g for 15 min; resuspended in ice-cold 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT; and lysed by Emulsiflex (Avestin). The resulting lysate was centrifuged at 100,000 ϫ g for 45 min, and GST-Ubc5 protein was purified from the 100,000 ϫ g supernatant by glutathione-Sepharose (GE Life Sciences) affinity chromatography using a 10-ml bed volume gravity flow column (40). The GST-E2 fusion proteins were processed with 50 units/ml thrombin (GE Life Sciences) to cleave the GST tag, and the resulting Ubc5 polypeptide was collected in the unadsorbed fraction of a subsequent glutathione-Sepharose affinity purification step designed to remove free GST.…”

Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

“…Interestingly, in experiments in which UAE was charged with NEDD8, a UBL that shares ϳ60% sequence homology with ubiquitin, it was shown that NEDD8 was efficiently transferred to UAE-ubiquitin pathway specific E2s (10,11). Furthermore, it was also found that E1 enzymes specifically bind their cognate E2 through specific interactions in their ␤-grasp domains irrespective of the UBL (12). This suggests that the UBL pathway specificity is determined by cognate recognition of UBL and E2 by the E1 and that there is no intrinsic discrimination against noncognate UBLs in E1-E2 transfer or subsequent steps (11).…”