Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase

Abstract

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2-CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2-CIB system by identifying versions of CRY2-CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13-α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

Figure 1. Truncations of of CRY2/CIB1 modules

(a) β-galactosidase activity of Gal4BD-CRY2 (full length and truncation constructs) and Gal4AD-CIB1 expressed in AH109/Y187 yeast and tested for interaction in the dark or blue light (1s pulse every 3 min, 461 nm, 5.8 mW/cm²) for 2.5 hrs. Data represent mean values ± s.e.m. (n=3 independent experiments) (b) β-galactosidase reporter activity of W303-1A yeast expressing different length LexABD-CRY2 constructs, VP16-CIB1, and a pSH18-34 reporter plasmid after 3 h treatment with blue light or dark. Data represent mean values ± s.e.m. for three independent experiments. (c) Fluorescence images of a HEK293T cell expressing CRY2PHR-mCh and membrane localized CIB81-pmEGFP (containing a CaaX motif at C-terminus of EGFP) pre and 1 min post blue light exposure. Graph depicted below shows quantification of the change in cytoplasmic CRY2PHR-mCh fluorescent signal over time after exposure to a single 488 nm pulse (mean values ± s.d., n=3 cells). Scale bar, 2 μm. (d) β-galactosidase activity of Gal4BD-CRY2 and indicated Gal4AD-CIB1 full length or truncation mutants expressed in AH109/Y187 yeast and tested for interaction in the dark or in blue light (1s pulse every 3 min, 461 nm, 5.8 mW/cm²) for 2.5 hrs. Data represent mean values of two independent experiments (3 replicates each).

Figure 2. Identification of CRY2 photocycle mutants

(a) Growth selection assay used for screening. Interaction of Gal4BD-CRY2(535) and Gal4AD-CIB1 results in reconstitution of Gal4 and activation of a URA3 reporter. (b) Schematic showing slow and fast cycling selection screens testing interaction of a GalBD-CRY2(535) mutant library with GalAD-CIB1. (c) Quantification of cytosolic levels of wild-type (wt) or indicated mutants in CRY2PHR-mCh upon recruitment to CIBN-pmEGFP at the plasma membrane after delivery of a pulse of 488nm light at time 0. The data represent mean values ± s.d., n=3 cells.

Figure 3. Sequence alignment and modeling of photocycle mutants

(a) Sequence alignment of CRYs from Arabidopsis thaliana (AtCRY1, AtCRY2), Ostreococcus tauri (OtCPF1, OtCPF2), Drosophila melanogaster (DmCRY), and Danaus plexippus (DpCRY). The amino acids resulting in photocycle alteration in AtCRY2 are indicated in red. Conserved hydrophobic residues and the residues involved in stabilizing electrostatic interactions are indicated in magenta and blue. (b) Location of mutations in AtCRY2 structure at end of helix α13. Key structural contacts are mediated by the α13-α14 turn motif (yellow helix-turn-helix) that positions conserved Trp353 adjacent to a salt bridge (Arg359-Asp387) required for signal transduction in DmCRY2. Arg357 of α14 forms a salt bridge to ANP in AtCRY1 crystal structures. Leu348 is buried in a hydrophobic pocket (Leu354, Ile358, Leu386) that anchors contacts between FAD, Arg359 (salt bridge, blue-dash) and Trp353, whereas Trp349 is partially solvent-exposed. The surrounding protein (grey) is transparent for clarity.

Figure 4. Functional analysis of CRY2 L348F

(a) CRY2 L348F allows extended transcriptional activation in response to a light pulse. Yeast expressing wt or L348F (also K329M) GalBD-CRY2(535) and Gal4AD-CIB1 were assayed for β-galactosidase reporter activity after 2.5 hrs in dark or under indicated light pulse conditions (1s pulse every 3 min, 461 nm, 5.8 mW/cm²). Data represent mean values ± s.d. (n=3 independent experiments) (b) Comparison of Cre recombinase activity generated using PA-Cre1.0 (CIBN-CreC/CRY2-CreN) or PA-Cre1.5 (same constructs, with L348F instead of wild-type CRY2). Constructs were transfected along with a fluorescent loxP-STOP-loxP-EGFP reporter in HEK293 cells, and exposed to a single 4s pulse of light 24 hr after transfection (461 nm, 5.8 mW/cm²). Reporter activity was quantified by flow cytometry 48 hours after transfection. Data represent mean values ± s.d. (n=3 independent experiments). *, p-value < .05 (c) Comparison of Cre recombinase activity of CRY2-CreN(wt or L348F) and CIB1-CreC(N1). Constructs were transfected and assayed as in (b). Data represent mean values of two independent experiments (triplicate replicates) analyzed by flow cytometry. Similar results were obtained by manual count in two independent experiments. (d) Cre recombinase activity of combined PA-Cre2.0 construct expressed in HEK293 cells. The combined PA-Cre2.0 construct was transfected along with a loxP-STOP-loxP-dsRed reporter, and exposed to a single 4s pulse of light 24 hr after transfection. Reporter activity was quantified by manual count 48 hrs after transfection. Data represent mean values ± s.d. (n=3) from one experiment, and experiments were repeated two times with similar results.