A DNA-based synthetic apoptosome

Abstract

Living cells are able to regulate key cellular processes by physically assembling signaling components on dedicated molecular platforms. The spatial organization of proteins in these higher-order signaling complexes facilitates proximity-driven activation and inhibition events, allowing tight regulation of the flow of information. Here, we employ the programmability and modularity of DNA origami as a controllable molecular platform for studying protein-protein interactions involved in intracellular signaling. Specifically, we engineer a synthetic, DNA origami-based version of the apoptosome, a large multi-protein signaling complex that regulates apoptosis by co-localization of multiple caspase-9 monomers. Our in vitro characterization using both wildtype caspase-9 monomers and inactive mutants tethered to a DNA origami platform directly demonstrates that enzymatic activity is induced by proximity-driven dimerization with asymmetric, half-of-sites reactivity. Additionally, experimental results supported by a detailed thermodynamic model reveal a multivalent activity enhancement in tethered caspase-9 oligomers of three and four enzymes, partly originating from a statistical increase in the number of active catalytic units in higherorder enzyme clusters. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami provides a modular platform to construct and characterize higher-order signaling complexes. The engineered DNA-based protein assembly platform has the potential to be broadly applied to inform the function of other important multi-enzyme assemblies involved in inflammation, innate immunity, and necrosis.

Dynamic modulation of proximity-induced enzyme activity using supramolecular polymers

“…Optimal enzyme activity was reached at equimolar amounts of both caspase-9 and DNA-Disc. Importantly, the nearly 5-fold increase in enzyme activity seen at 20 nM of DNA-Disc agrees with the enhancement seen in previous studies where caspase-9 was dimerized on a DNA origami-based nanoscaffold at roughly the same caspase-9 concentration, 34 or with other in vitro systems that typically do not reach the activity of apoptosome activated caspase-9. [31][32][33]35 A further increase in DNA-Disc concentration, beyond the optimum, diminished enzyme activity ( Fig.…”

“…[31][32][33] In the present system, the CARD domain has been replaced by a DNA strand, that recruits caspase-9 onto the nanoscaffold through DNA-duplex formation. 34 The catalytic domain was expressed with an N-terminal noncanonical amino acid (p-azidophenylalanine), introduced by using amber codon suppression in E. coli, in order to site-specifically decorate the protein with the DNA anti-handle (ESI †). 34 The DNA contains a 5 0 -end nucleotide linker between the enzyme and the recruitment recognition sequence (anti-handle) in order to avoid undesired interactions with the nanoscaffold.…”

“…34 The catalytic domain was expressed with an N-terminal noncanonical amino acid (p-azidophenylalanine), introduced by using amber codon suppression in E. coli, in order to site-specifically decorate the protein with the DNA anti-handle (ESI †). 34 The DNA contains a 5 0 -end nucleotide linker between the enzyme and the recruitment recognition sequence (anti-handle) in order to avoid undesired interactions with the nanoscaffold. A detailed quantitative analysis has previously demonstrated that the kinetic characteristics of the caspase-9 enzyme-DNA conjugate is similar to wildtype caspase-9.…”

“…A detailed quantitative analysis has previously demonstrated that the kinetic characteristics of the caspase-9 enzyme-DNA conjugate is similar to wildtype caspase-9. 34 The modularity and dynamic nature of monomer intermixing in the supramolecular nanoscaffold allows straightforward tuning of the nanoscaffold characteristics, such as the systematic variation of the recruiter handle density in the nanoscaffold ( Fig. 1c and d), which potentially regulates enzymatic activity through the variation of inter-enzyme distances, as seen in natural systems.…”

“…A series of control experiments were carried out to rule out possible unspecific interactions between the components in the system. A constant concentration of caspase-9 (25 nM, well below its dimerization affinity 34 ) was incubated with non-DNA-functionalized monomers (Inert-Disc) at different concentrations ( Fig. S1, ESI †).…”