Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

Pankaj Kumar, Pearl Magala, Kathryn R. Geiger-Schuller, Ananya Majumdar, Joel R. Tolman, Cynthia Wolberger

Research output: Contribution to journalArticle

Abstract

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.

Original languageEnglish (US)
Pages (from-to)9039-9050
Number of pages12
JournalNucleic Acids Research
Volume43
Issue number18
DOIs
StatePublished - 2015

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Ubiquitin
Histones
Mutation
Ubiquitin-Conjugating Enzymes
Catalytic Domain
Yeasts
Enzymes

ASJC Scopus subject areas

  • Genetics

Cite this

Kumar, P., Magala, P., Geiger-Schuller, K. R., Majumdar, A., Tolman, J. R., & Wolberger, C. (2015). Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity. Nucleic Acids Research, 43(18), 9039-9050. https://doi.org/10.1093/nar/gkv845

Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity. / Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia.

In: Nucleic Acids Research, Vol. 43, No. 18, 2015, p. 9039-9050.

Research output: Contribution to journalArticle

Kumar, P, Magala, P, Geiger-Schuller, KR, Majumdar, A, Tolman, JR & Wolberger, C 2015, 'Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity', Nucleic Acids Research, vol. 43, no. 18, pp. 9039-9050. https://doi.org/10.1093/nar/gkv845
Kumar, Pankaj ; Magala, Pearl ; Geiger-Schuller, Kathryn R. ; Majumdar, Ananya ; Tolman, Joel R. ; Wolberger, Cynthia. / Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity. In: Nucleic Acids Research. 2015 ; Vol. 43, No. 18. pp. 9039-9050.
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