Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximityinduced 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 multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype 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 additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The DNA origami technique has proven to have tremendous potential for therapeutic and diagnostic applications like drug delivery, but the relatively low concentrations of cations in physiological fluids cause destabilization and degradation of DNA origami constructs preventing in vivo applications. To reveal the mechanisms behind DNA origami stabilization by cations, we performed atomistic molecular dynamics simulations of a DNA origami rectangle in aqueous solvent with varying concentrations of magnesium and sodium as well as polyamines like oligolysine and spermine. We explored the binding of these ions to DNA origami in detail and found that the mechanism of stabilization differs between ion types considerably. While sodium binds weakly and quickly exchanges with the solvent, magnesium and spermine bind close to the origami with spermine also located in between helices, stabilizing the crossovers characteristic for DNA origami and reducing repulsion of parallel helices. In contrast, oligolysine of length ten prevents helix repulsion by binding to adjacent helices with its flexible side chains, spanning the gap between the helices. Shorter oligolysine molecules with four subunits are weak stabilizers as they lack both the ability to connect helices and to prevent helix repulsion. This work thus shows how the binding modes of ions influence the stabilization of DNA origami nanostructures on a molecular level.

Cellular signaling is regulated by the assembly of proteins into higher‐order complexes. Bottom‐up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild‐type symmetric CT32–CT32 interface of the 14‐3‐3–CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14‐3‐3 scaffold. Biochemical studies, supported by mass‐balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher‐order protein complexes. Enzyme complementation on the 14‐3‐3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.

Cellular signaling is regulated by the assembly of proteins into higher‐order complexes. Bottom‐up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild‐type symmetric CT32–CT32 interface of the 14‐3‐3–CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14‐3‐3 scaffold. Biochemical studies, supported by mass‐balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher‐order protein complexes. Enzyme complementation on the 14‐3‐3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions.

the accurate mechanism of how EIIC transports a sugar has not been clearly demonstrated yet. To investigate more detailed and accurate transportation mechanisms, we performed collective variable-based steered molecular dynamics (CVSMD) simulations. Our simulation shows the spontaneous transportation of the sugar toward the opposite side of the membrane with the preserved H-bonding interactions, supporting the ''Elevator'' type transport mechanism. A set of cross-linking experiments was carried out on the basis of the CVSMD model structures, and we were able to acquire a few of stable cross-linked structures.