Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2016 Aug 2;113(31):8705-10. doi: 10.1073/pnas.1524648113. Epub 2016 Jul 19.

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

Author information

1
Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1;
2
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
3
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel jshifman@mail.huji.ac.il sachdev.sidhu@utoronto.ca.
4
Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1; jshifman@mail.huji.ac.il sachdev.sidhu@utoronto.ca.

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.

KEYWORDS:

USP; hot spots; phage display; saturation scanning; ubiquitin

PMID:
27436899
PMCID:
PMC4978272
DOI:
10.1073/pnas.1524648113
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center