Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway Journal Article
| Authors: | Yunus, A. A.; Lima, C. D. |
| Article Title: | Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway |
| Abstract: | E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members. © 2006 Nature Publishing Group. |
| Keywords: | unclassified drug; mutation; mutant protein; ubiquitin; chemical analysis; protein conformation; allele; protein; wild type; protein p53; prediction; cloning, molecular; amino acid sequence; sequence homology, amino acid; kinetics; substrate specificity; crystallography, x-ray; catalysis; structure analysis; sumo protein; sumo-1 protein; ubiquitin-conjugating enzymes; mutagenesis; lysine; genetic complementation; x ray analysis; conjugation; protein ubc9; nup358 protein; ranbp2 protein |
| Journal Title: | Nature Structural and Molecular Biology |
| Volume: | 13 |
| Issue: | 6 |
| ISSN: | 1545-9993 |
| Publisher: | Nature Publishing Group |
| Date Published: | 2006-06-26 |
| Start Page: | 491 |
| End Page: | 499 |
| Language: | English |
| DOI: | 10.1038/nsmb1104 |
| PUBMED: | 16732283 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 54" - "Export Date: 4 June 2012" - "CODEN: NSMBC" - "Source: Scopus" |
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