Designing photoswitchable peptides using the AsLOV2 domain

Abstract

Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Jα helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49- and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light-dependent colocalization, which we demonstrate with photo-activable gene transcription in yeast.

Figure 1. General design strategy for caging peptides using the AsLOV2 domain

Photoswitches are designed as sequence chimeras between the AsLOV2 Jα helix and the peptide to be caged. Residues that are important to AsLOV2-Jα interactions (cyan), important to peptide-target interaction (purple), important to both interactions (red), and residues that are important to neither interaction (white) are identified and mutated accordingly. Irradiation unfolds the Jα helix, and the peptide can bind its target.

Figure 2. Design of LOV-ipaA and LOV-SsrA peptide photoswitches

(A) Sequence alignment of AsLOV2-Joc, ipaA, and LOV-ipaA. Jα sequence (blue) ipaA sequence (purple), chimera sequence (cyan), and designed residues (red) are indicated. (B) Sequence alignment of AsLOV2-Jα helix, SsrA peptide, and 3 LOV-SsrA designs, LOV-SsrAC, LOV-SsrAN, and LOV-SsrAM. Jα helix sequence (blue), SsrA sequence (orange), chimera sequence (cyan), designed positions (black), and helix stabilizing mutations (pink) are indicated. (C) Model of LOV-ipaA with residues colored as in (A). Residues N538, I539, A542, A543 K544 (cyan) as well as residues N537, K541, D545, V546 (purple) and I540 (red) are shown as sticks. (D) Model of LOV-SsrAC with residues colored as in (B). Residues A528,E538 (pink) as well as residues A542, A543 (cyan) and N544, D545, E546, N547, Y548 (orange) are shown as sticks. See also Figure S1.

Figure 3. Measurement of rates and affinities of LOV-ipaA binding to vinculin

(A) Schematic of fluorescence polarization competition assay is shown. TAMRA labeled ipaA (ipaA*) is bound to vinculinD1 subdomain (vinD1). Vinculin dissociates from the complex with rates k₁, k₁ and binding affinity K_D1. LOV-ipaA (LOVipaA) binds vinculin with rates k_on, k_off and affinity K_D2. Fluorescence polarization decreases as the fraction of TAMRA-ipaA bound to vinculin decreases. (B) Fraction of TAMRA-ipaA bound to vinculin over time with varying concentrations of LOV-ipaA titrated in the dark and (C) under blue light. (D) Surface plasmon resonance measurements and first-order binding fit of LOV-ipaA L514K L531E C450A pseudo-dark and (E) LOV-ipaA L514K L531E A532E I53E pseudo-lit mutants. See also Figure S2.

Figure 4. Competitve binding of LOV-SsrA to SspB in blue light and in darkness

Competitive binding assay of LOV-SsrAC (A) or LOV-SsrAN (B) to an equilibrium solution of SspB and 5(6)TAMRA-SsrA. 5(6)TAMRA-SsrA becomes unbound as LOV-SsrA competes for SspB binding. Binding to SspB was measured immediately after illumination with blue light (open circles) and after return to dark state (closed circles). (C) Reversible binding of LOV-SsrAC to SspB. A single titration point from the fluorescence polarization competition assay was repeatedly irradiated with blue light (blue bar = 60 seconds) and reversion to dark state equilibrium was monitored by polarization. See also Figure S3 and Table S1.

Figure 5. Binding of LOV-ipaA to full-length vinculin is measured through an actin co-sedimentation assay

(A) Full-length vinculin, LOV-ipaA, and polymerized actin are incubated 1 hr at room temperature. Vinculin that is bound to LOV-ipaA will bind polymerized actin. The mixture is centrifuged at 150,000 g, pelleting polymerized actin and all vinculin bound to it out of solution. (B) SDS-page gel of LOV-ipaA C450A and (C) LOV-ipaA A532E I536E actin co-sedimentation assay with vinculin. Molar ratios from 1:0 to 1:50 vinculin:LOV-ipaA were used. Supernatant (S) and pellet (P) fractions are shown side by side. Apparent binding affinity curves of fraction of vinculin bound to actin v. concentration of LOV-ipaA are plotted below. See also Table S2.

Figure 6. LOV-ipaA is used as a light-inducible heterodimerization tool

(A) LOV-ipaA L623A is linked to the GAL4 activation domain (AD), while vinculinD1 (vinD1) is linked to GAL4 binding domain (BD). Irradiation with blue light brings AD-LOV-ipaA into proximity to BD-vinD1, allowing for GAL-induced transcription of reporter genes LacZ, MEL1, HIS2, and ADE2. (B) LacZ transcription is quantified. β-galactosidase activity of S. cerevisiae mated strains containing BD and AD linked proteins, as specified, is shown. (C) S. cerevisiae mated strains containing BD-vinD1 and AD-LOV-ipaA mutants, as indicated, are grown in dark or blue light conditions on SD plates. Difference in levels of transcription of MEL1, HIS3 and ADE2 in dark v. lit state conditions is seen. See also Figure S4.