Directed evolution of ligand dependence: small-molecule-activated protein splicing

Abstract

Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.

Fig. 2.

Characterization of initial evolved inteins. (A) Yeast cultures expressing clones 1-14 and 2-4 inserted into GFP at position 108 were grown for 24 h in the presence or absence of 10 μM 4-HT and analyzed by flow cytometry. The bimodal fluorescence distribution arises from loss of the GFP-encoding plasmid in the less fluorescent cells as revealed by their inability to grow on medium selective for the presence of the plasmid. (B) Protein splicing in clones 1-14, 2-4, and 2-5 was evaluated by Western blot analysis with an anti-GFP antibody after growth for 24 h in the absence (–) or presence (+) of 10 μM 4-HT. Splicing removes the intein–ER fusion from the 74-kDa precursor (upper band) to yield the 27-kDa GFP (lower band).

Fig. 3.

Evolved ligand-dependent intein 3-2 modulates protein function in living cells in four contexts. (A) FACS analysis of yeast expressing intein 3-2 inserted into GFP after 24 h of growth in the presence or absence of 4-HT. The gene encoding the intein–GFP construct was integrated into yeast genomic DNA to preclude loss of the construct. (B) Intein 3-2 inserted into the KanR context was plated on medium containing 100 μg/ml geneticin in the presence or absence of 4-HT. (C) Yeast expressing LacZ containing intein 3-2 were grown 16 h with or without 4-HT and assayed for β-galactosidase activity (20) in triplicate. LacZ activities are normalized relative to the same yeast strain lacking the lacZ gene. (D) Yeast expressing intein 3-2 inserted into the endogenous S. cerevisiae protein Ade2p were plated on medium containing 10 mg/liter adenine in the presence or absence of 4-HT and compared with an ade2^– strain. Yeast lacking Ade2p activity accumulate a red pigment; colonies with Ade2p activity maintain their white color.

Fig. 4.

Properties of evolved intein 3-2. (A) Genomically integrated GFP disrupted with intein 3-2 (intein 3-2–GFP) was expressed in the absence of 4-HT for 16 h to accumulate unspliced protein. One hour after cycloheximide (100 μg/ml) was added to inhibit new protein synthesis, 10 μM 4-HT was added, and aliquots were frozen rapidly at various time points to evaluate splicing kinetics by Western blot analysis (see Fig. 2B). (B) Yeast expressing intein 3-2–GFP were grown with varying concentrations of 4-HT for 28 h and analyzed by flow cytometry to determine the dose dependence of intein activity. (C) Lanes: 1–4, yeast expressing intein 3-2–GFP were grown 20 h in the absence or presence of 10 μM GDA and in the absence or presence of 10 μM 4-HT and then evaluated by Western blot; 5–7, in a separate experiment, yeast expressing intein 3-2–GFP were grown 16 h to accumulate prespliced protein and then treated with 100 μg/ml cycloheximide for 1 h followed by 10 μM GDA. Western blot analysis was performed on aliquots frozen 0, 2, and 6 h after GDA treatment.

Fig. 1.

Approach and early results described in this work. (A) Strategy for the directed evolution of a ligand-dependent intein (see text for details). (B) Intein–ER fusion clones 1-1 and 1-14 were inserted into the kanamycin-resistance protein (KanR) at position 119. S. cerevisiae cells expressing these constructs were plated on medium containing 150 μg/ml geneticin in the presence or absence of 10 μM 4-HT.

Fig. 5.

Mutations responsible for evolved ligand dependence. (Left) Intein mutations in the minimal 3-2 clone (Ala-34 → Val, blue; and His-41 → Leu, pink) are mapped onto the homologous Ssp DnaB mini-intein structure (30). (Right) The location of acquired ER-LBD mutations of minimal clone 3-2 (Val-376 → Ala, yellow; Arg-521 → Gly, red) are mapped onto the ER-LBD structure, with helix 12 in orange and bound 4-HT in black (22). Images were rendered with pymol.