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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The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request.
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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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).
The authors declare no competing interests.
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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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