title: Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.
abstract: Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).
authors.affiliation: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.|School of Chemistry, University Park, University of Nottingham, Nottingham, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA.
authors.forename: Tom P
authors.initials: TP
authors.lastname: Garner
authors.affiliation: School of Chemistry, University Park, University of Nottingham, Nottingham, UK.|Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK.
authors.forename: Jed
authors.initials: J
authors.lastname: Long
authors.affiliation: Institute of Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Jo
authors.initials: J
authors.lastname: Strachan
authors.affiliation: Protein & Nucleic Acid Chemistry Laboratory, University of Leicester, Leicester, UK.
authors.forename: Sharad C
authors.initials: SC
authors.lastname: Mistry
authors.affiliation: Protein & Nucleic Acid Chemistry Laboratory, University of Leicester, Leicester, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
authors.affiliation: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Tooth
authors.affiliation: School of Chemistry, University Park, University of Nottingham, Nottingham, UK.|Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.affiliation: School of Chemistry, University Park, University of Nottingham, Nottingham, UK.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
authors.affiliation: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
authors.forename: Rob
authors.initials: R
authors.lastname: Layfield
mesh_terms: D001665:Binding Sites; D001681:Biological Assay / Q000592:standards*; D045424:Complex Mixtures / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D000072417:Protein Domains; D015202:Protein Engineering; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D054875:Ubiquitination
chemical_list: D045424:Complex Mixtures; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; D008239:Lysine
keywords: Native mass spectrometry; Protein engineering; Technology; Ubiquitin-binding domain; Ubiquitination; Unanchored polyubiquitin