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

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 fa...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2016-08, Vol.113 (31), p.8705-8710
Main Authors: Leung, Isabel, Dekel, Ayelet, Shifman, Julia M., Sidhu, Sachdev S.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding sites
Binding Sites - genetics
Biochemistry
Biological Sciences
Computer Simulation
Endopeptidases - chemistry
Endopeptidases - genetics
Endopeptidases - metabolism
Enzymes
Epitopes - chemistry
Epitopes - genetics
Epitopes - metabolism
Humans
Kinetics
Models, Molecular
Molecular chemistry
Mutagenesis
Proteases
Protein Binding
Protein Domains
Proteins
Sequence Homology, Amino Acid
Ubiquitin - chemistry
Ubiquitin - genetics
Ubiquitin - metabolism
Ubiquitin Thiolesterase - chemistry
Ubiquitin Thiolesterase - genetics
Ubiquitin Thiolesterase - metabolism
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1524648113