A versatile strategy for the semisynthetic production of Ser65 phosphorylated ubiquitin and its biochemical and structural characterisation

Cong Han, Kuan-Chuan Pao, Agne Kazlauskaite, Miratul M. K. Muqit, Satpal Virdee (Lead / Corresponding author)

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

Original languageEnglish
Pages (from-to)1574-1579
Number of pages6
JournalChemBioChem
Volume16
Issue number11
DOIs
Publication statusPublished - 27 Jul 2015

Keywords

  • Enzyme catalysis
  • Ligases
  • Phosphorylation
  • Synthetic methods
  • Ubiquitin

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    Student Theses

    Design and Synthesis of an E3 ligase Activity-Based Probe and its Application for the Discovery of a New Class of E3 Ligase

    Author: Pao, K., 2018

    Supervisor: Virdee, S. (Supervisor)

    Student thesis: Doctoral ThesisDoctor of Philosophy

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    Regulation of the Parkin E3 ligase by PINK1-dependent phosphorylation

    Author: Kazlauskaite, A., 2015

    Supervisor: Muqit, M. (Supervisor) & Alessi, D. (Supervisor)

    Student thesis: Doctoral ThesisDoctor of Philosophy

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    Virdee, Satpal

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