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Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin
Authors:Daniel Scott  Tom P Garner  Jed Long  Jo Strachan  Sharad C Mistry  Andrew R Bottrill  David J Tooth  Mark S Searle  Neil J Oldham  Rob Layfield
Institution:1. School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK;2. School of Chemistry, University Park, University of Nottingham, Nottingham, UK;3. Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA;4. Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK;5. Institute of Cell Biology, University of Edinburgh, Edinburgh, UK;6. Protein & Nucleic Acid Chemistry Laboratory, University of Leicester, Leicester, UK
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 ).
Keywords:Native mass spectrometry  Protein engineering  Technology  Ubiquitination  Ubiquitin‐binding domain  Unanchored polyubiquitin
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