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Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Jennifer A. Miles, Fruzsina Hobor, James Taylor, Christian Tiede, Philip R. Rowell, Chi H. Trinh, Brian Jackson, Fatima Nadat, Hannah F. Kyle, Basile I. M. Wicky, Jane Clarke, Darren C. Tomlinson, Andrew J. Wilson, View ORCID ProfileThomas A. Edwards
doi: https://doi.org/10.1101/651364
Jennifer A. Miles
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Fruzsina Hobor
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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James Taylor
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Christian Tiede
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Philip R. Rowell
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Chi H. Trinh
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Brian Jackson
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
dProtein Production Facility, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Fatima Nadat
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
dProtein Production Facility, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Hannah F. Kyle
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Basile I. M. Wicky
eDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW
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Jane Clarke
eDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW
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Darren C. Tomlinson
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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Andrew J. Wilson
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
cSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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  • For correspondence: T.A.Edwards@leeds.ac.uk A.J.Wilson@leeds.ac.uk
Thomas A. Edwards
aSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
bAstbury Centre For Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
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  • ORCID record for Thomas A. Edwards
  • For correspondence: T.A.Edwards@leeds.ac.uk A.J.Wilson@leeds.ac.uk
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Abstract

The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-of-concept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications.

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Posted May 27, 2019.
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Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs
Jennifer A. Miles, Fruzsina Hobor, James Taylor, Christian Tiede, Philip R. Rowell, Chi H. Trinh, Brian Jackson, Fatima Nadat, Hannah F. Kyle, Basile I. M. Wicky, Jane Clarke, Darren C. Tomlinson, Andrew J. Wilson, Thomas A. Edwards
bioRxiv 651364; doi: https://doi.org/10.1101/651364
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Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs
Jennifer A. Miles, Fruzsina Hobor, James Taylor, Christian Tiede, Philip R. Rowell, Chi H. Trinh, Brian Jackson, Fatima Nadat, Hannah F. Kyle, Basile I. M. Wicky, Jane Clarke, Darren C. Tomlinson, Andrew J. Wilson, Thomas A. Edwards
bioRxiv 651364; doi: https://doi.org/10.1101/651364

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