Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation

Kalman P. Bencsath, Michael S. Podgorski, Vishwajeeth R. Pagala, Clive A. Slaughter, Brenda A. Schulman

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.

Original languageEnglish (US)
Pages (from-to)47938-47945
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number49
DOIs
StatePublished - Dec 6 2002
Externally publishedYes

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Ubiquitins
Binding Sites
Adenosine Triphosphate
Enzymes
Ubiquitin
Ligation
Saccharomyces cerevisiae
Yeast
Mutation
Chemical activation
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation. / Bencsath, Kalman P.; Podgorski, Michael S.; Pagala, Vishwajeeth R.; Slaughter, Clive A.; Schulman, Brenda A.

In: Journal of Biological Chemistry, Vol. 277, No. 49, 06.12.2002, p. 47938-47945.

Research output: Contribution to journalArticle

Bencsath, Kalman P. ; Podgorski, Michael S. ; Pagala, Vishwajeeth R. ; Slaughter, Clive A. ; Schulman, Brenda A. / Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 49. pp. 47938-47945.
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