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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