De novo design of allosterically driven disassembly of protein complexes

Abstract

Protein design has focused on the design of ground states, ensuring they are sufficiently low energy to be highly populated. Designing the kinetics and dynamics of a system requires, in addition, the design of excited states that are traversed in transitions from one low-lying state to another. This is a challenging task as such states must be sufficiently strained to be poorly populated, but not so strained that they are not populated at all, and because protein design methods have mainly focused on creating near-ideal structures.We developed a general approach for designing systems that use an induced-fit forcing motion to generate a strained excited state, allosterically driving protein complex dissociation in a process termed “facilitated dissociation.” Kinetic binding measurements demonstrate that incorporating excited states enables design of effector-induced acceleration of dissociation as high as 5,700-fold. Crystal structures throughout the facilitated dissociation process demonstrate accurate design of such excited states. Within these excited states, we find that strain distributes nonuniformly depending on local topology, leading to kinetic asymmetry. Kinetic binding measurements with multiple effectors show that flexibility facilitates the force-generating conformational transition used to rapidly form the strained excited state. We further investigated whether facilitated dissociation could enable faster-timescale control over cellular processes. Many cytokines form tight complexes with their receptors that dissociate or degrade on timescales of hours, so controlling the temporal dynamics of signaling is difficult: there is no off switch. We designed an off-switchable IL-2 mimic, enabling seconds-timescale control over its signaling, and used this tool to investigate early events in IL-2 signaling. This also demonstrated the generality of our approach: in principle, by fusing to our switch, almost any binder can be made to rapidly dissociate in the presence of effector. Overall, this work provides a route to designing the rates and pathways of protein motion and change, which should ultimately enable construction of complex lifelike protein machines demanding precise timing and rapid functional change.