title: A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.
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.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).
authors.forename: Cong
authors.initials: C
authors.lastname: Han
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).
authors.forename: Kuan-Chuan
authors.initials: KC
authors.lastname: Pao
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).
authors.forename: Agne
authors.initials: A
authors.lastname: Kazlauskaite
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).
authors.forename: Miratul M K
authors.initials: MM
authors.lastname: Muqit
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK). s.s.virdee@dundee.ac.uk.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
mesh_terms: D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D008958:Models, Molecular; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D012694:Serine / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D004220:Disulfides; D010750:Phosphoproteins; D025801:Ubiquitin; D012694:Serine; D044767:Ubiquitin-Protein Ligases
keywords: enzyme catalysis; ligases; phosphorylation; synthetic methods; ubiquitin