Designed asymmetric protein assembly on a symmetric scaffold

Cellular signaling is regulated by assembly of proteins into higher-order complexes. Bottom-up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Here, we present the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. We targeted the wildtype symmetric CT32-CT32 interface of the 14-3-3/CT32 complex, ultimately favoring asymmetric assembly on the 14-3-3 scaffold. Biochemical studies, supported by mass-balance models, allowed for 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.