Identification and characterization of mutations in ubiquitin required for non-covalent dimer formation

Gabrielsen, M., Buetow, L., Kowalczyk, D., Zhang, W., Sidhu, S. S. and Huang, D. T. (2019) Identification and characterization of mutations in ubiquitin required for non-covalent dimer formation. Structure, 27(9), 1452-1459.e4. (doi: 10.1016/j.str.2019.06.008) (PMID:31303481) (PMCID:PMC6720194)

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Abstract

Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein domains makes Ub an attractive scaffold for engineering ubiquitin variants (UbVs) with high target specificity. Previously, we identified a UbV that formed a non-covalent stable dimer via a β-strand exchange, and in the current work we identified and characterized the minimal substitutions in the primary sequence of Ub required to form a higher ordered complex. Using solution angle scattering and X-ray crystallography, we show that a single substitution of residue Gly10 to either Ala or Val is sufficient to convert Ub from a monomer to a dimer. We also investigate contributions to dimer formation by the residues in the surrounding sequence. These results can be used to develop next-generation phage-display libraries of UbVs to engineer new interfaces for protein recognition.

Item Type:Articles
Additional Information:The authors thank W. Clark and A. Keith for in-house DNA sequencing, and Diamond Light Source for access to stations I04-1 and B21 (BAG allocation MX16258). This work was supported by Cancer Research UK (A23278) and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no 647849) awarded to D.T.H. and a Genome Canada Disruptive Innovation in Genomics grant (OGI-119) and a CQDM-OCE EXPLORE Project (#23927) awarded to S.S.S. W.Z. was supported by a Cancer Research Society/ BMO Bank of Montreal Scholarship for the Next Generation of Scientists.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Kowalczyk, Dominika and Huang, Professor Danny and Buetow, Dr Lori and Gabrielsen, Dr Mads
Authors: Gabrielsen, M., Buetow, L., Kowalczyk, D., Zhang, W., Sidhu, S. S., and Huang, D. T.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Journal Name:Structure
Publisher:Elsevier (Cell Press)
ISSN:0969-2126
ISSN (Online):1878-4186
Published Online:11 July 2019
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in Structure 27(9): 1452-1459.e4
Publisher Policy:Reproduced under a Creative Commons License

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