Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

Scott, Daniel; Garner, Tom P.; Long, Jed; Strachan, Jo; Mistry, Sharad C.; Bottrill, Andrew R.; Tooth, David J.; Searle, Mark S.; Oldham, Neil J.; Layfield, Rob

Abstract

Unanchored polyubiquitin chains are emerging as importanregulators 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 described. We describe the design of a linker-optimised 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. Utilising 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 favour 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 non-biological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the ‘ubiquitome’.

Citation

Scott, D., Garner, T. P., Long, J., Strachan, J., Mistry, S. C., Bottrill, A. R., Tooth, D. J., Searle, M. S., Oldham, N. J., & Layfield, R. (in press). Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin. Proteomics, 16(14), https://doi.org/10.1002/pmic.201600067

• Journal Article Type: Article
• Acceptance Date: Mar 29, 2016
• Online Publication Date: Jul 21, 2016
• Deposit Date: Apr 4, 2016
• Publicly Available Date: Jul 21, 2016
• Journal: Proteomics
• Print ISSN: 1615-9853
• Electronic ISSN: 1615-9861
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 16
• Issue: 14
• DOI: https://doi.org/10.1002/pmic.201600067
• Public URL: https://nottingham-repository.worktribe.com/output/800077
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1002/pmic.201600067/abstract
• Additional Information: "This is the peer reviewed version of the following article: Scott, Daniel and Garner, Tom P. and Long, Jed and Strachan, Jo and Mistry, Sharad C. and Bottrill, Andrew R. and Tooth, David J. and Searle, Mark S. and Oldham, Neil J. and Layfield, Rob (2016) Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin. Proteomics. ISSN 1615-9853, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/pmic.201600067/abstract. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
• Contract Date: Apr 4, 2016