De novo design of allosterically switchable protein assemblies

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central role in the control of metabolism and cell signaling 1–3 . There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such “action at a distance” modulation and to create synthetic proteins whose functions can be regulated by effectors 4–7 . However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge 8,9 . Here, inspired by the classic Monod-Changeux-Wyman model of cooperativity 10 , we investigate the de novo design of allostery through rigid-body coupling of designed effector-switchable hinge modules 11 to protein interfaces 12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to effector binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry 13 , and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of effectors and can have ligand-binding cooperativity comparable to classic natural systems such as hemoglobin 14 . Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized sidechain-sidechain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines, and cellular feedback control circuitry.