Photoswitchable binders enable temporal dissection of endogenous protein function.

General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.

Determinants for intrinsically disordered protein recruitment into phase-separated protein condensates.

Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often biased residues of IDPs are also likely involved in selective recruitment of many other IDPs into condensates. However, determining factors for this IDP recruitment into protein condensates are not understood yet. Here, we quantitatively examined recruitment tendencies of various IDPs with different sequence compositions into IDP-clustered condensates both in vitro as well as in cells. Condensate-forming IDP scaffolds, recruited IDP clients, and phase separation conditions were carefully varied to find key factors for selective IDP partitioning in protein condensates. Regardless of scaffold sequences, charged residues in client IDPs assured potent IDP recruitment, likely via strong electrostatic interactions, where positive residues could further enhance recruitment, possibly with cation-pi interactions. Notably, poly-ethylene glycol, a widely used crowding reagent for in vitro phase separation, abnormally increased IDP recruitment, indicating the need for careful use of crowding conditions. Tyrosines of IDP clients also strongly participated in recruitment both in vitro and in cells. Lastly, we measured recruitment degrees by more conventional interactions between folded proteins instead of disordered proteins. Surprisingly, recruitment forces by an even moderate protein interaction (K d ∼ 5 µM) were substantially stronger than those by natural IDP-IDP interactions. The present data offer valuable information on how cells might organize protein partitioning on various protein condensates.

Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions.

Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid-liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

Behavior control of membrane-less protein liquid condensates with metal ion-induced phase separation.

Phase separation of specific biomolecules into liquid droplet-like condensates is a key mechanism to form membrane-less organelles, which spatio-temporally organize diverse biochemical processes in cells. To investigate the working principles of these biomolecular condensates as dynamic reaction centers, precise control of diverse condensate properties is essential. Here, we design a strategy for metal ion-induced clustering of minimal protein modules to produce liquid protein condensates, the properties of which can be widely varied by simple manipulation of the protein clustering systems. The droplet forming-minimal module contains only a single receptor protein and a binding ligand peptide with a hexahistidine tag for divalent metal ion-mediated clustering. A wide range of protein condensate properties such as droplet forming tendency, droplet morphology, inside protein diffusivity, protein recruitment, and droplet density can be varied by adjusting the nature of receptor/ligand pairs or used metal ions, metal/protein ratios, incubation time, binding motif variation on recruited proteins, and even spacing between receptor/ligand pairs and the hexahistidine tag. We also demonstrate metal-ion-induced protein phase separation in cells. The present phase separation strategy provides highly versatile protein condensates, which will greatly facilitate investigation of molecular and structural codes of droplet-forming proteins and the monitoring of biomolecular behaviors inside diverse protein condensates.

Homo-molecular Fluorescence Complementation for Direct Visualization of Receptor Oligomerization in Living Cells.

Cell surface receptor oligomerization is an attractive target process for drug screening. However, simple but reliable (and thus high-throughput) visualization methods for receptor oligomerization are still lacking. Herein, we report on a new single-construct homo-molecular fluorescence complementation (Homo-FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal-to-noise ratio was not affected by expression level variations of fused receptors. The Homo-FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo-FC reliably visualized the oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI.

Discovery of fluorescent 3-heteroarylcoumarin derivatives as novel inhibitors of anaplastic lymphoma kinase.

Altered expression or hyperactivation of anaplastic lymphoma kinase (ALK), as a consequence of translocations or point mutations, is one of the main oncogenic drivers in non-small cell lung cancer. Using structure-based design and in vitro enzyme assays, we identified 3-heteroarylcoumarin as a new template for the development of novel fluorescent ALK inhibitors. Molecular simulation provided structural insights for the design of 3-heteroarylcoumarin derivatives, which were easily prepared through efficient synthetic approaches including direct C-H cross coupling. Importantly, these coumarin-based ALK inhibitors can be tracked using microscopy techniques: we illustrated the use of the most potent compound in this series, 5a, (ALK/IC50 = 0.51 µM, λemi = 500 nm, φF = 0.29) to monitor its subcellular distribution pattern by confocal fluorescence microscopy.