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Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering

Nature Chemical Biology volume 17, pages 915–923 (2021)Cite this article

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Abstract

Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR–Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.

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Fig. 1: Design of photoswitchable LOV2 circular permutants (cpLOV2).
Fig. 2: cpLOV2 affords new surface areas to cage protein activity.
Fig. 3: Design of a cpLOV2-based optogenetic suicide device.
Fig. 4: Photoswitchable CAR T cells for inducible tumor cell killing.
Fig. 5: Wireless optogenetic control of CAR T cell proliferation and tumor killing in vivo.

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Data availability

Supplementary data are available online. The plasmids and reagents are available from the corresponding authors on reasonable request. Key constructs used in the study were also deposited in the nonprofit plasmid repository Addgene (nos. 168991–168996). Source data are provided with this paper.

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Acknowledgements

This work was supported by the Welch Foundation (grant no. BE-1913-20190330 to Y.Z.) and the American Cancer Society (grant nos. RSG-16-215-01-TBE to Y.Z. and RSG-18-043-01-LIB to Y.H.). All NMR experiments were performed at High Magnetic Field Laboratory of the Chinese Academy of Sciences. We thank S.K. Siwko at Institute of Biosciences and Technology, Texas A&M University for his constructive criticism of the paper.

Author information

Author notes

  1. These authors contributed equally: Lian He, Peng Tan, Lei Zhu, Kai Huang.

Authors and Affiliations

  1. Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA

    Lian He, Peng Tan, Nhung T. Nguyen, Rui Wang, Lei Guo, Zixian Huang & Yubin Zhou

  2. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Peng Tan

  3. High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China

    Lei Zhu, Ling Li & Junfeng Wang

  4. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Worcester, MA, USA

    Kai Huang & Gang Han

  5. Center for Epigenetics and Disease Prevention, Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX, USA

    Yuhan Yang & Yun Huang

  6. Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui, China

    Junfeng Wang

  7. Department of Translational Medical Sciences, College of Medicine, Texas A&M University, Houston, TX, USA

    Yubin Zhou

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Contributions

Y.Z. and Y.H. conceived the ideas. Y.Z., Y.H., G.H. and J.W. directed the work. Y.Z., L.H., P.T., G.H., K.H., J.W. and L.Z. designed the study. L.H., P.T., L.Z. and N.T.N. performed the cellular experiments. L.H., P.T. and R.W. performed in vivo experiments. L.G. performed viral packaging experiment. L.Z. and L.L. purified the protein and performed in vitro biophysical characterization. L.H., L.Z., N.T.N. and P.T. analyzed the results. Z.H. and Y.Y. carried out the immunoblotting and T7E1 experiments. K.H. and G.H. provided the nanomaterials. Y.Z., L.H., L.Z. and P.T. wrote the paper. All of the authors contributed to the discussion and editing of the paper.

Corresponding authors

Correspondence to Yun Huang, Gang Han, Junfeng Wang or Yubin Zhou.

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Competing interests

Y.Z. and G.H. have submitted a patent application to the United States Patent and Trademark Office pertaining to the design and biomedical applications aspect(s) of this work (application number 62/942,770). The remaining authors declare no competing interests.

Additional information

Peer review information Nature Chemical Biology thanks Dominik Niopek, Yingxiao Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 cpLOV2 design.

a, (Left) The original N- and C-termini (black spheres) of LOV2 are separated by 22.7 Å. The locations of newly created N-termini (magenta spheres) were mapped to the crystal structure of LOV2. (Right) 2D topology view of LOV2. Permutation sites to create new N/C termini are indicated with magenta circles. b, Reversible cpLOV2-Zdk2 interaction following five repeated light-dark cycles (t1/2, activation = 9.8 ± 0.8 s; t1/2, deactivation = 57.4 ± 3.1 s; n = 12 cells from three independent biological replicates; mean ± s.e.m.). c, The deactivation kinetics of cpLOV2 mutants upon withdrawal of blue light illumination. Mutations were introduced into cpLOV2 to diversify the photo-responsive kinetics. Q513L caused a notable delay in the deactivation half-life (t1/2, off: 494 s versus 54 s of WT cpLOV2), whereas I427V accelerated the deactivation process by over 2-fold (t1/2, deactivation = 24 s). n = 24 cells for WT, n = 21 cells for I427V, n = 22 cells for Q513L; three independent biological replicates were performed. Data are shown as mean ± s.e.m.

Source data

Extended Data Fig. 2 Optimization of cpLOV2.

a, Generation of truncated Jα-cpLOV2 constructs to map minimal structural elements required to maintain light-inducible association between cpLOV2 and Zdk2. The presence of at least a partial Jα helix was needed to mediate effective cpLOV2-Zdk2 association. Truncation until 525 was tolerated without pronounced loss of the magnitude of response; but further truncations significantly compromised light-induced changes (n = 20 cells from three independent biological replicates; mean ± s.e.m.). b, Mutations in cpLOV2 (left) and Zdk2 (right) to tune the binding strength of the light-inducible interaction. The locations of introduced mutations were mapped to the 3D structure of the LOV2-Zdk2 complex (PDB entry: 5DJT). n = 30–37 cells from three independent biological replicates (mean ± s.e.m.).

Source data

Extended Data Fig. 3 Light-induced structural changes in cpLOV2 revealed by high-resolution NMR.

a, The secondary structure of cpLOV2 in the dark state was predicted based on the backbone chemical shifts. Probability values > 0.7 or < −0.7 (horizontal dashed lines) indicates significant changes from random coil. Shown on the top is the secondary structure of LOV2. b, Schematic (left panel) and a representative photograph (right panel) illustrating the installation of an external LED to enable acquisition of the NMR spectrum for cpLOV2 in its blue light-illuminated state. c, 2D 1H-15N HSQC spectra of cpLOV2 in the dark (black) and lit states (red). The conformation of cpLOV2 was greatly altered after blue light illumination as reflected by the massive changes in chemical shifts of multiple peaks. d, Overlaid 2D 1H-15N HSQC spectra of LOV2 (black) and cpLOV2 (red) in the dark (left) or lit states (right). The conformation of cpLOV2 was very similar to LOV2 in the lit state. e, Surface representation of cpLOV2. Circle, new surface areas (left, 3.7 nm2; right, 7.4 nm2) that can be utilized to cage an effector domain fused to the N terminus of cpLOV2. The new N- and C- termini are shown in red and blue, respectively.

Extended Data Fig. 4 cpLOV2-Zdk2 association revealed by high-resolution NMR and ITC.

a, Thermodynamic parameters derived from ITC data shown in Fig. 1d. b, Comparison of dissociation constants (Kd) between LOV2-Zdk2 and cpLOV2-Zdk2 interactions (n = 3 independent biological replicates; mean ± s.e.m.). c,d, 2D 1H-15N HSQC spectra of cpLOV2 (c) or Zdk2 (d) in free (black) and bound (red) states demonstrated their physical interaction in solution. e, The amide CSP (Δδ, ppm) of cpLOV2 upon Zdk2 binding. CSPs were calculated by the equation Δδ = [(ΔδHN)2 + (0.17ΔδN)2]0.5 and plotted versus residue numbers. Mean (0.23 ppm), Mean + SD (0.54 ppm) and Mean + 2 SD (0.85 ppm) values of Δδ are indicated by dot, dash, and solid lines, respectively. Asterisks indicate prolines or residues with no data. See Fig. 1e for the mapping of affected residues to the modeled structure of cpLOV2.

Extended Data Fig. 5 cpLOV2 affords new surface areas to cage protein activity.

Engineered STIM1-mediated activation of the ORAI Ca2+ channel was utilized to test the caging capability. Light-induced Ca2+ influx in HeLa cells was monitored by GCaMP6m. To create light-operated Ca2+ channel actuators (LOCCa), two effector domains (SOAR-I, STIM1336–486 or SOAR-II, STIM1344–442) were incorporated into LOV2 or cpLOV2 as indicated, in the presence or absence of Zdk2. Samples sizes: n = 25 and 30 cells for V1(left and right bars) from three independent biological replicates; V2, 49 and 50; V3, 25 and 25; V4, 42 and 25; V5, 25 and 25; V6, 27 and 61; V7, 31 and 25; V8, 46 and 46. Data are shown as mean ± s.e.m. Fmax, maximal fluorescent signals with photostimulation; F0, basal fluorescent signals in the dark.

Extended Data Fig. 6 Grafting cpLOV2 into AcrIIA4 to create a light-switchable anti-Cas9 inhibitor (LiCASINO).

a, The 3D structure of the spCas9-sgRNA-AcrIIA4 complex. Sphere, cpLOV2 insertion sites. b, The domain organization of AcrIIA4-cpLOV2 hybrids. V5 showed the most prominent light-dependent activity. Linkers (GGSG or GGSGG) used to flank the grafted cpLOV2 into the host protein were indicated. c, Light-inducible Cas9-inhibitory activity of LiCASINO. (left) Cartoon depicting the design of the screening assay. HEK293T cells were co-transfected with dCas9-VPR, the indicated AcrIIA4-cpLOV2 variants, and an sgRNA targeted to a synthetic promoter upstream of GFP. In the dark, LiCASINO tightly binds dCas9 to prevent its binding to the target genomic locus. Upon light irradiation, cpLOV2 alters the conformation of AcrIIA4 to diminish its dCas9-inhibitory activity, thereby restoring the function of dCas9-VPR to turn on GFP expression. (right) Quantification of light-inducible changes in GFP expression (n = 3 independent biological replicates; mean ± s.e.m.). VPR, a synthetic transcriptional coactivator made of VP64, p65 and Rta. P values are determined by unpaired two-tailed Student’s t-test. d, T7E1 assay to confirm photo-controllable genome editing at an endogenous genomic locus. HEK293T cells were co-transfected with Cas9, LiCASINO, and an sgRNA designed to disrupt the endogenous CCR5 gene. e, Immunoblot analysis of the effect of light on the interaction between FLAG-Cas9 and LiCASINO-mCh2. HEK293T cells were co-transfected with both constructs. FLAG-Cas9 was immobilized on anti-FLAG beads as the bait to pulldown the indicated AcrIIA4 variants. WT AcrIIA4 was used as a positive control and GAPDH was used as loading control.

Extended Data Fig. 7 LOV2-MLKL-N did not induce light-dependent necroptosis.

a, Domain architectures of the LOV2 or cpLOV2-based cell suicide constructs. b, Time-lapse confocal imaging of HeLa cells expressing mCh-LOV2-MLKL-N. SYTOX blue is used as an indicator of dead cells. Scale bar, 20 μm.

Extended Data Fig. 8 cpLOV2-based optical dimerizer to photo-control protein localization and subcellular events.

a, Photo-triggered interaction between PM-anchored ssrA–cpLOV2-CAAX and mCh-sspB fused to effector domains (5-ptase for PI(4,5)P2 to PI4P conversion; or iSH2 of PI3K for (PI(4,5)P2 to PI(3,4,5)P3 conversion) in HeLa cells. These manipulations allowed light-inducible reprogramming of phosphoinositide metabolism in the plasma membrane (PM). GFP-PHPLCδ and PHAKT-GFP were used as PI(4,5)P2 and PI(3,4,5)P3 sensors, respectively. Scale bar, 10 µm. b, cpLOV2 enables light-inducible protein diffusion into the primary cilia of NIH3T3 cells. (Left) a schematic depicting light-inducible recruitment of ssrA-cpLOV2-GFP into the cilia (Arl13b-mCh-sspB as the bait). (Right) representative confocal images of NIH3T3 cells co-expressing ssrA-cpLOV2-GFP (green) and Arl13b-mCh-sspB (red) before and after blue light illumination for 2 min. Scale bar, 10 µm. Also see Supplementary Video 5. c, Light-inducible recruitment of targets to the nuclear envelope. sspB-mEmerald-Lamin A (green) was anchored to the nuclear envelope and ssrA-cpLOV2-mCh-NES (red) remained smoothly distributed within the nucleoplasm in the dark. Upon photostimulation, ssrA-cpLOV2-mCh-NES translocated to the nuclear envelope. Scale bar, 10 µm. Also see Supplementary Video 6. d, Bioluminescence to report light-inducible transcriptional activation mediated by dCas9-ssrA-cpLOV2 and sspB-VPR. (left) Cartoon illustrating the design of photo-induced association of dCas9-ssrA-cpLOV2 with sspB-VPR to turn on luciferase reporter gene expression. (right) Quantification of luciferase activity in HEK293T cells co-expressing the indicated constructs (n = 3 independent biological replicates; mean ± s.e.m.).

Extended Data Fig. 9 Design and optimization of photoswitchable optoCAR constructs.

a, Design of split CAR candidate constructs to enable light-inducible assembly of intact chimeric antigen receptors (optoCARs). The optical heterodimerization domains ssrA-cpLOV2/LOV2-ssrA and sspB are included in Part A and Part B, respectively. The A1 and B2 construct highlighted in red showed the best performance. B5 without the T cell activation domain CD3ζ was constructed as negative control. b, Light-inducible recruitment of Part B (red) to the PM-resident Part A (green) visualized in Jurkat T cells. Scale bar, 5 μm. c, NFAT-dependent luciferase expression in Jurkat T cells transduced with the indicated combinations of optoCAR components. Jurkat T cells were transduced with viruses encoding WT CAR or optoCARs (A1 + B combinations shown in panel a) and co-cultured with CD19+ Raji cells. Cell were either shielded or exposed to pulsed blue light illumination overnight. T cell activation was measured by the activity of the Ca2+/NFAT-dependent luciferase reporter gene (n = 3 independent biological replicates; mean ± s.e.m.). d, Immunoblot analysis of cell lysates from Jurkat T cells to confirm the expression of each CAR component. Antibodies against GFP and mCherry were used to probe the expression of Part A and Part B, respectively. e, Flow cytometry analysis of cell surface CD69 expression in engineered human CD4+ T cells. Under dark (gray) or lit (blue) states, human primary CD4+ T cells were transduced with retroviruses expressing WT CAR, optoCAR, or defective CAR constructs, and co-cultured with CD19-negative K562 cells or CD19-positive Raji cells.

Extended Data Fig. 10 Comparison of in vitro tumor killing efficiency mediated by variable CARs.

a, Human CD8+ T cells expressing WT CAR, ON-switch CAR, optoCAR, or LINTAD CAR were co-cultured with Raji tumor cells (20 hours) at a T cell:tumor cell (E/T) ratio of 40:1. ON-switch CAR cells were treated with or without 500 nM rapalog (AP21967). OptoCAR cells were kept in the dark or stimulated with blue light (pulse light, 5 s ON + 1 min OFF). LINTAD CAR cells were pre-treated with blue light to stimulate CAR expression (1 s ON + 29 s OFF for 12 hours), followed by co-culture with Raji tumor cells. n = 3 independent biological replicates (mean ± s.e.m.). Unpaired two-tailed Student’s t-test was performed. b, CD19+ Raji cell viability measured by the MTT assay when incubated with different concentrations of rapalog (AP21967), which was used in the ON-stitch CAR system. n = 6 independent biological replicates (mean ± s.e.m.). Adjusted P values were determined by one-way ANOVA with Sidak’s multiple comparisons test.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Light-induced mitochondria-to-cytosol dissociation of cpLOV2-mCh2 in HeLa cells cotransfected with mitochondria-anchored Zdk2. Three repeated dark–light cycles (470 nm, 4 mW cm−2) were applied to visualize the reversible cpLOV2–Zdk2 dissociation (light) and association (dark). Related to Fig. 1b and Extended Data Fig. 1b.

Supplementary Video 2

Light-induced calcium influx in HeLa cells expressing a LOCCa ON-switch variant (LOCCa-V9.1). Calcium levels were reported by a genetically encoded red color calcium indicator jRCaMP1b (false color using blue-to-red scale; blue, low calcium; green/red, high calcium). Three dark–light cycles were applied. Related to Fig. 2a.

Supplementary Video 3

Light-induced suppression of calcium influx in HeLa cells expressing a LOCCa OFF-switch variant (LOCCa-V10.1). Calcium levels were monitored by jRCaMP1b (false color using blue-to-red scale; blue, low calcium; green/red, high calcium). Three dark–light cycles were applied. Related to Fig. 2c.

Supplementary Video 4

Time-lapse imaging of light-triggered necroptosis in live HeLa cells. HeLa cells expressing MLKL-N-cpLOV2-mCh2 were stimulated with pulsed blue light (470 nm light, 4 mW cm−2, cycle of 1 min ON and 4 min OFF). Blue, dead cell stained with SYTOX blue. Related to Fig. 3b.

Supplementary Video 5

Light-inducible recruitment of cytosolic ssrA–cpLOV2-GFP (green) into the primary cilium (indicated by the coexpressed Arl13b-mCh-sspB; red) in an NIH3T3 cell. Images were acquired every 6 s for 2 min. Related to Extended Data Fig. 8b.

Supplementary Video 6

Blue light-triggered ssrA–cpLOV2-NLS-mCh2 shuttling between the nucleoplasm and the nuclear envelop in HeLa cells. sspB-mEmerald-Lamin A was coexpressed and two light–dark cycles were applied. Related to Extended Data Fig. 8c.

Additional Supplementary Files 1

Source data for Supplementary Fig. 6.

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He, L., Tan, P., Zhu, L. et al. Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering. Nat Chem Biol 17, 915–923 (2021). https://doi.org/10.1038/s41589-021-00792-9

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