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][1]–[3][2]. 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][3]–[7][4]. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge[8][5],[9][6]. Here, inspired by the classic Monod-Changeux-Wyman model of cooperativity[10][7], we investigate the de novo design of allostery through rigid-body coupling of designed effector-switchable hinge modules[11][8] to protein interfaces[12][9] 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][10], 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][11]. 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. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-7 [5]: #ref-8 [6]: #ref-9 [7]: #ref-10 [8]: #ref-11 [9]: #ref-12 [10]: #ref-13 [11]: #ref-14