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Specific targeting of human caspases using designed ankyrin repeat proteins

Andreas Flütsch, Thilo Schroeder, Jonas Barandun, Rafael Ackermann, Martin Bühlmann and Markus G. Grütter
From the journal Biological Chemistry

Abstract

Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1–9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.


Corresponding author: Markus G. Grütter, Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, e-mail:
aThese authors contributed equally to this work.bPresent address: University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0629, USA.cPresent address: Nextech Invest Ltd., Scheuchzerstrasse 35, 8006 Zurich, Switzerland.dPresent address: The Rockefeller University, Laboratory of Protein and Nucleic Acid Chemistry, 1230 York Avenue, New York, NY 10065, USA.

Acknowledgments

Financial support of this work was provided by the Swiss National Science Foundation grant 310030-122342 to M.G.G. We thank Beat Blattmann and Céline Stutz-Ducommun from the NCCR crystallization facility for crystal screening and the staff of the X06SA beamline at the Swiss Light Source of the Paul Scherrer Institute (PSI) for their support during data collection. Dr. Christopher Weinert is acknowledged for his calibration data of the size exclusion column.

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Supplemental Material

The online version of this article (DOI: 10.1515/hsz-2014-0173) offers supplementary material, available to authorized users.

Received: 2014-3-24
Accepted: 2014-8-5
Published Online: 2014-9-5
Published in Print: 2014-10-1

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