Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21

Isabel Leung, Ayelet Dekel, Julia M. Shifman*, Sachdev S. Sidhu

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Original languageAmerican English
Pages (from-to)8705-8710
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number31
DOIs
StatePublished - 2 Aug 2016

Bibliographical note

Funding Information:
Canadian Cancer Society (Grant 702861).

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