Specific targeting of human caspases using designed ankyrin repeat proteins

Andreas Flütsch 1 , Thilo Schroeder 1 , Jonas Barandun 1 , Rafael Ackermann 1 , Martin Bühlmann 1  and Markus G. Grütter 1
  • 1 Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Andreas Flütsch, Thilo Schroeder, Jonas Barandun, Rafael Ackermann, Martin Bühlmann and Markus G. Grütter


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.

    • Supplemental_Data
  • Amstutz, P., Binz, H.K., Parizek, P., Stumpp, M.T., Kohl, A., Grütter, M.G., Forrer, P., and Plückthun, A. (2005). Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715–24722.

  • Binz, H.K., Stumpp, M.T., Forrer, P., Amstutz, P., and Plückthun, A. (2003). Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332, 489–503.

  • Binz, H.K., Amstutz, P., Kohl, A., Stumpp, M.T., Briand, C., Forrer, P., Grütter, M.G., and Plückthun, A. (2004). High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575–582.

  • Blanchard, H., Kodandapani, L., Mittl, P.R., Marco, S.D., Krebs, J.F., Wu, J.C., Tomaselli, K.J., and Grütter, M.G. (1999). The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis. Structure 7, 1125–1133.

  • Boucher, D., Blais, V., and Denault, J.B. (2012). Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis. Proc. Natl. Acad. Sci. USA 109, 5669–5674.

  • Bravman, T., Bronner, V., Lavie, K., Notcovich, A., Papalia, G.A., and Myszka, D.G. (2006). Exploring “one-shot” kinetics and small molecule analysis using the ProteOn XPR36 array biosensor. Anal. Biochem. 358, 281–288.

  • Duarte, J.M., Srebniak, A., Schärer, M.A., and Capitani, G. (2012). Protein interface classification by evolutionary analysis. BMC Bioinform. 13, 334.

  • Favaloro, B., Allocati, N., Graziano, V., Di Ilio, C., and De Laurenzi, V. (2012). Role of apoptosis in disease. Aging 4, 330–349.

  • Flütsch, A., Ackermann, R., Schroeder, T., Lukarska, M., Hausammann, G.J., Weinert, C., Briand, C., and Grütter, M.G. (2014). Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise. Biochem. J. 461, 279–290.

  • Fuentes-Prior, P. and Salvesen, G.S. (2004). The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384, 201–232.

  • Grütter, M.G. (2000). Caspases: key players in programmed cell death. Curr. Opin. Struct. Biol. 10, 649–655.

  • Hanes, J. and Plückthun, A. (1997). In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942.

  • Hardy, J.A., Lam, J., Nguyen, J.T., O’Brien, T., and Wells, J.A. (2004). Discovery of an allosteric site in the caspases. Proc. Natl. Acad. Sci. USA 101, 12461–12466.

  • Kabsch, W. (2010). Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144.

  • McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674.

  • McStay, G.P., Salvesen, G.S., and Green, D.R. (2008). Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15, 322–331.

  • Merz, T., Wetzel, S.K., Firbank, S., Plückthun, A., Grütter, M.G., and Mittl, P.R. (2008). Stabilizing ionic interactions in a full-consensus ankyrin repeat protein. J. Mol. Biol. 376, 232–240.

  • Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.

  • Rohn, T.T. (2010). The role of caspases in Alzheimer’s disease; potential novel therapeutic opportunities. Apoptosis 15, 1403–1409.

  • Roschitzki-Voser, H., Schroeder, T., Lenherr, E.D., Frölich, F., Schweizer, A., Donepudi, M., Ganesan, R., Mittl, P.R., Baici, A., and Grütter, M.G. (2012). Human caspases in vitro: expression, purification and kinetic characterization. Protein Expr. Purif. 84, 236–246.

  • Salvesen, G.S. and Dixit, V.M. (1999). Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10964–10967.

  • Schroeder, T., Barandun, J., Flütsch, A., Briand, C., Mittl, P.R., and Grütter, M.G. (2013). Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors. Structure 21, 277–289.

  • Schweizer, A., Roschitzki-Voser, H., Amstutz, P., Briand, C., Gulotti-Georgieva, M., Prenosil, E., Binz, H.K., Capitani, G., Baici, A., Plückthun, A., et al. (2007). Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. Structure 15, 625–636.

  • Seeger, M.A., Zbinden, R., Flütsch, A., Gutte, P.G., Engeler, S., Roschitzki-Voser, H., and Grütter, M.G. (2013). Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity. Protein Sci. 22, 1239–1257.

  • Sennhauser, G., Amstutz, P., Briand, C., Storchenegger, O., and Grütter, M.G. (2007). Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, e7.

  • Stefan, N., Martin-Killias, P., Wyss-Stoeckle, S., Honegger, A., Zangemeister-Wittke, U., and Plückthun, A. (2011). DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J. Mol. Biol. 413, 826–843.

  • Steiner, D., Forrer, P., and Plückthun, A. (2008). Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J. Mol. Biol. 382, 1211–1227.

  • Studier, F.W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234.

  • Taylor, R.C., Cullen, S.P., and Martin, S.J. (2008). Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241.

  • Watt, W., Koeplinger, K.A., Mildner, A.M., Heinrikson, R.L., Tomasselli, A.G., and Watenpaugh, K.D. (1999). The atomic-resolution structure of human caspase-8, a key activator of apoptosis. Structure 7, 1135–1143.

  • Zahnd, C., Amstutz, P., and Plückthun, A. (2007). Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269–279.

Purchase article
Get instant unlimited access to the article.
Log in
Already have access? Please log in.

Journal + Issues

Biological Chemistry keeps you up-to-date with the latest advances in the molecular life sciences. The journal publishes Research Articles, Short Communications, Reviews and Minireviews. Areas include: general biochemistry/pathobiochemistry, structural biology, molecular and cellular biology, genetics and epigenetics, virology, molecular medicine, plant molecular biology/biochemistry and novel experimental methodologies.