Proximity-induced caspase-9 activation on a DNA origami-based synthetic apoptosome

Abstract

Living cells regulate key cellular processes by spatial organization of catalytically active proteins in higher-order signalling complexes. These act as organizing centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein–protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multiprotein complex that regulates apoptosis by colocalizing multiple caspase-9 monomers. Tethering of both wild-type and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity and, furthermore, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.

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Fig. 1: General concept and design elements for the construction of a DNA-based synthetic apoptosome.
Fig. 2: Characterization of caspase-9 assembly onto DNA origami nanostructures.
Fig. 3: Activation of caspase-9 occurs by distance-dependent dimerization of tethered monomers.
Fig. 4: Colocalization of more than two caspase-9 monomers leads to enhanced enzymatic activity.
Fig. 5: Enzymatic activity of the caspase-9 dimer originates from a single catalytic site.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request.

Code availability

Custom-written code for the computer models and simulations that support the experimental findings in this study is available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. van Dongen for help with the mass spectrometry analyses, N. van der Zon for initial protein expression experiments and G. Cremers for helpful discussions. The ICMS Animation Studio contributed the cartoons of DNA strands and the DNA origami structure. This work was supported by the European Research Council (project no. 677313 BioCircuit), an NWO-VIDI grant from the Netherlands Organisation for Scientific Research (723.016.003) and funding from the Ministry of Education, Culture and Science (Gravity programmes 024.001.035 and 024.003.013).

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B.J.H.M.R. designed the study, performed experiments, developed the geometric model, analysed the data, and wrote the manuscript. A.J.M. developed and derived the thermodynamic model and analysed the data. B.G.A. performed and analysed all AFM measurements. J.A.L.R. performed molecular dynamics simulations. A.d.H. performed initial protein expression and provided critical input for the experiments. L.B. supervised the study and provided critical feedback on the manuscript. T.F.A.d.G. conceived, designed and supervised the study, analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Luc Brunsveld or Tom F. A. de Greef.

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Rosier, B.J.H.M., Markvoort, A.J., Gumí Audenis, B. et al. Proximity-induced caspase-9 activation on a DNA origami-based synthetic apoptosome. Nat Catal 3, 295–306 (2020). https://doi.org/10.1038/s41929-019-0403-7

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