A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases

Maryna Gorelik; Noah Manczyk; Alevtina Pavlenco; Igor Kurinov; Sachdev S Sidhu; Frank Sicheri

doi:10.1016/j.str.2018.06.004

A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases

Structure. 2018 Sep 4;26(9):1226-1236.e3. doi: 10.1016/j.str.2018.06.004. Epub 2018 Jul 19.

Authors

Maryna Gorelik¹, Noah Manczyk², Alevtina Pavlenco³, Igor Kurinov⁴, Sachdev S Sidhu⁵, Frank Sicheri⁶

Affiliations

¹ Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
² Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
³ Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada.
⁴ Department of Chemistry and Chemical Biology, Cornell University, Argonne, IL 60439, USA.
⁵ Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: sachdev.sidhu@utoronto.ca.
⁶ Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: sicheri@lunenfeld.ca.

Abstract

Skp1-Cul1-F-box (SCF) E3 ligases constitute the largest and best-characterized family of the multisubunit E3 ligases with important cellular functions and numerous disease links. The specificity of an SCF E3 ligase is established by one of the 69 human F-box proteins that are recruited to Cul1 through the Skp1 adaptor. We previously reported generation of ubiquitin variants (UbVs) targeting Fbw7 and Fbw11, which inhibit ligase activity by binding at the F-box-Skp1 interface to competitively displace Cul1. In the present study, we employed an optimized engineering strategy to generate specific binding UbVs against 17 additional Skp1-F-box complexes. We validated our design strategy and uncovered the structural basis of binding specificity by crystallographic analyses of representative UbVs bound to Skp1-Fbl10 and Skp1-Fbl11. Our study highlights the power of combining phage display with structure-based design to develop UbVs targeting specific protein surfaces.

Keywords: E3 ligases; Fbl10; Fbl11; Kdm2a; Kdm2b; X-ray crystallography; phage display; structural biology; ubiquitin variants; ubiquitination.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Binding Sites
Cell Surface Display Techniques
Crystallography, X-Ray
Cullin Proteins / metabolism
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology
F-Box Proteins / metabolism
Female
HEK293 Cells
Humans
Jumonji Domain-Containing Histone Demethylases / metabolism
Models, Molecular
Protein Binding
SKP Cullin F-Box Protein Ligases / antagonists & inhibitors
SKP Cullin F-Box Protein Ligases / chemistry*
SKP Cullin F-Box Protein Ligases / metabolism*
Small Molecule Libraries / chemistry
Small Molecule Libraries / pharmacology
Structure-Activity Relationship
Ubiquitins / chemistry*
Ubiquitins / pharmacology*

Substances

Cullin 1
Cullin Proteins
Enzyme Inhibitors
F-Box Proteins
Small Molecule Libraries
Ubiquitins
Jumonji Domain-Containing Histone Demethylases
KDM2A protein, human
SKP Cullin F-Box Protein Ligases

Abstract

Publication types

MeSH terms

Substances

Grants and funding