Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here we use light or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into non-conserved, surface exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, GTPases, guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, and modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.

Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.spatiotemporal control | cell motility | endothelial-mesenchymal transition T he past two decades have seen a revolution in computational protein design, with remarkable milestones including design of a helical protein from first principles (1), redesign of zinc finger proteins (2), and de novo design of an α/β protein (3). These studies highlighted, as a proof of principle, our ability to rationally control the structure of proteins by using basic physical principles and phenomenology. These approaches are based on finding an optimal sequence for a given single structure or ensemble of related states, and do not provide a strategy to construct a protein capable of large on-demand conformational transitions (4, 5). A number of multistate protein design algorithms (4, 6) have been proposed; however, designing an experimentally confirmed, regulatable multistate protein, or a conformational switch (5), still remains as a challenging task because of the necessity of engineering and controlling multiple protein states (4,7,8).Such a conformational switch protein has great advantages in cell signaling, because it can be used as a universal regulatory domain (9) for precise, specific, and temporal control over rapidly activated signaling proteins (5, 10-15). Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5, 13). Recently developed protein switches, including derivatives of the light, oxygen, or voltage (LOV) domain (16, 17), can provide direct control at the protein level with light, but cannot be readily used in nontransparent animals. Our previous rapamycin regulated (RapR) kinase method (14) can potentially overcome this problem, but it requires expression and control of two proteins. The variable stoichiometry of these proteins renders the response more heterogeneous and essentially impractical in animals. Therefore, a single-chain, insertable, and transferable regulatory domain would be very valuable.Here we design a ligand-controlled conformational switch, uniRapR, a potentially broadly applicable, single-chai...

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites.

Behavioral experiences activate the Fos transcription factor (TF) in sparse populations of neurons that are critical for encoding and recalling specific events 1 – 3 . However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos -activated cells. It is also unknown if Fos is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos -expressing hippocampal CA1 pyramidal neurons by parvalbumin (PV)-interneurons (INs) is enhanced, while perisomatic inhibition by cholecystokinin (CCK)-INs is weakened. This bidirectional modulation of inhibition is abolished when the function of the Fos TF complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling, and chromatin analyses, combined with electrophysiology, reveal that Fos activates the transcription of Scg2 (secretogranin II), a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As PV- and CCK-INs mediate distinct features of pyramidal cell activity 4 – 6 , the Scg2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo . Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to CA1 theta are significantly altered with loss of Scg2 . These findings reveal an instructive role for Fos and Scg2 in establishing a network of Fos -activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms on distinct inhibitory pathways may support the consolidation of memories over time.