Genetically Engineered Photoinducible Homodimerization System

Jan 15, 2014 - Clackson , T., Yang , W., Rozamus , L. W., Hatada , M., Amara , J. F., Rollins , C. T., Stevenson , L. F., Magari , S. R., Wood , S. A...
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Genetically Engineered Photoinducible Homodimerization System with Improved Dimer-Forming Efficiency Yuta Nihongaki, Hideyuki Suzuki, Fuun Kawano, and Moritoshi Sato* Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan S Supporting Information *

ABSTRACT: Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a lightinducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.

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provides a unique photoinducible dimerization system based on homodimerization. Most other dimerization systems are based on heterodimerization of photoreceptors and their binding proteins. Homodimerzation is an important mechanism for managing cellular signaling and governs the activation of various key signaling proteins, such as membrane receptors, kinases, proteases, and transcription factors.12 To optically control these proteins, which are activated through homodimerization, a photoinducible homodimerization system is superior to a heterodimerization system. Taken together, VVD could be a promising photoinducible homodimerization system to optically control various molecular processes in the cell. Despite the above advantages over other photoinducible dimerization systems, VVD has a critical limitation. The problem is that the binding affinity of VVD in the light state is low (Kd = 13 μM),13 which causes low efficiency of homodimer formation. In contrast, in the rapamycin-inducible protein−protein interaction system based on FK506 binding protein (FKBP) and the FKBP12-rapamycin binding protein (FRB),14 a representative chemically inducible dimerization (CID) system, the dissociation constant between the two protein in the presence of rapamycin is 12 nM.15 This characteristic has made CID systems robust and versatile for controlling diverse cellular signaling pathways. In the history of developing FKBP-based homodimer system, improving the affinity of this system by engineering protein and its chemical

hotoactivation of cellular proteins has recently emerged as a powerful strategy for understanding complex biological systems based on precise spatial and temporal coordination of signaling events.1,2 In the field of chemical biology, many technologies for photoactivation of proteins have been developed, such as synthesis of caged peptides and the genetic insertion of photocaged unnatural amino acids.2 Recently, another approach to photoactivation of proteins, called optogenetics, has appeared.1,3 The outstanding characteristic of optogenetics tools is genetic encodability with natural amino acids, which comes from natural photoreceptors derived from bacteria, algae, fungus, and plants.3 Recently, several groups have reported photoinducible protein−protein interaction systems based on natural photoreceptors.4−9 These systems have pioneered optical control of various cellular signaling processes. Vivid (VVD) is a photoreceptor derived from Neurospora crassa and rapidly forms a homodimer in response to blue light.10 VVD has several advantages over other photoinducible dimerization systems. First, VVD (150 amino acids) is much smaller than any other dimerization systems based on photoreceptors, such as PhyB (908 amino acids)-PIFs (PIF3, 211 amino acids and PIF6, 100 amino acids),4 CRY2 (612 amino acids)-CIB1 (355 amino acids),5 and FKF1-LOV (167 amino acids)-truncated GI (391 amino acids).7,11 This beneficial property of VVD allows us to avoid occurrence of a steric hindrance and greatly facilitate accurate molecular design of optogenetic tools. Second, VVD functions in mammalian cells without addition of an exogenous cofactor, because the cofactor of VVD is flavin adenine dinucleotide (FAD), which abundantly exists in eukaryote cells. Lastly, VVD © 2014 American Chemical Society

Received: November 10, 2013 Accepted: January 15, 2014 Published: January 15, 2014 617

dx.doi.org/10.1021/cb400836k | ACS Chem. Biol. 2014, 9, 617−621

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To examine the involvement of a disulfide bond in the enhancement of the homodimerization activity by the I52C mutation, we performed the bioluminescence assay under strongly reducing environment using 10 mM dithiothreitol (DTT) and found that the DTT treatment did not decrease the enhanced homodimerization activity of VVD-52C (Figure 2b). We also examined an effect of the I52C mutation on the dissociation kinetics of the homodimer. Because the homodimer of VVD has a long lifetime to dissociate due to its long photocycle (time constant τ = 18,000 s),20 we compared the dissociation kinetics of a fast-photocycle mutant of VVD having I85V substitution (VVD-85V) and a mutant having both I85V and I52C substitutions (VVD-52C85V) by the bioluminescence assay (Figure 2c). We induced homodimerization of VVD-85V and that of VVD-52C85V upon blue light irradiation and then observed their dissociation after turning off the irradiation. While VVD-52C85V exhibited a dimerization activity higher than that of VVD-85V (Supplementary Figure 3), no significant difference was observed in the dissociation kinetics between VVD-85V and VVD-52C85V (Figure 2c). We further carried out Western blot analysis and observed no significant difference of expression level between VVD and VVD-52C (Figure 2d). These results underpin that the I52C mutation actually enhances the binding affinity of VVD-52C but the enhancement of homodimerization is not necessarily due to the stable disulfide bond formation between the cysteine 52 residues. Next, we applied I52C mutation to VVD-56K71V, which are known to reduce the background activity of VVD.8 In our bioluminescence assay, VVD-56K71V showed slightly lower background activity compared to VVD, although VVD-56K71V also showed lower dimerization activity in the light state (Supplementary Figure 4). We confirmed that the I52C mutation enhanced the dimer-forming activity of VVD56K71V at both light and dark state conditions (Supplementary Figure 4). VVD-52C56K71V showed lower dimer-forming activity in the light state compared to that of VVD-52C. The fold increase of VVD-52C and VVD-52C56K71V are almost the same, 9.1-fold and 7.6-fold, respectively. There have also been several reported variants of VVD, represented by VVD-71V, which is characterized to have higher dimer-forming efficiency in vitro.13 However, VVD-71V did not show higher dimer-forming efficiency in living cells (Supplementary Figure 4). Therefore, the presented 52C mutation is the first example that can enhance dimer-forming efficiency of VVD in vivo. We applied VVD-52C to recently reported light-inducible gene expression system, termed the LightOn system.8 The LightOn system has led to experiments on oscillatory control of transcription factor.21 This system is based on a genetically encoded light-switchable transactivator, called GAVP, which consists of three functional domains, the Gal4 DNA-binding domain, VVD, and the p65 transactivation domain (Figure 3a). The GAVP transactivator binds the upstream activating sequence of Gal (UAS) upon blue light irradiation and initiates transcription of target genes (Figure 3b). We compared the light-induced reporter gene expression between GAVP and the transactivator equipped with VVD-52C (GAVP-52C) (Figure 3c). These transactivators exhibited light-dependent reporter gene expression. GAVP-52C showed 9.1-fold greater lightinduced expression of luciferase compared with GAVP. For further characterization of VVD-52C, GAVP and GAVP-52C were expressed with a minimal enhancerless SV40 (ΔSV40)

ligand significantly contributed to make a chemically inducible homodimerization system robust.16,17 In consideration of this fact, it is important to enhance the affinity of VVD homodimer in the light state. In the present study, we conduct protein engineering of VVD to improve its homodimer-forming efficiency and thereby develop a potent photoinducible homodimerization system. According to the crystal structure of the light-state dimer of VVD, the N-terminal α helix contributes to the interface through the contact of an exposed hydrophobic face that involves Ile52 (Figure 1).18 The side chain of Ile52 is in close

Figure 1. Crystal structure of VVD dimer in the light state. The two VVD are colored in cyan and blue, respectively. Ile52 is represented by stick model and highlighted in yellow. FAD is depicted in red. Enlarged image of N-terminal α helix contact is also shown.

proximity to the same position of the other VVD protein in the light state. To enhance the dimer-forming efficiency of VVD, we performed site-directed saturation mutagenesis of Ile52. We assessed the dimer-forming efficiency of these I52X mutants of VVD by a bioluminescence assay based on complementation of split luciferase fragments.19 To conduct this assay, we constructed fusion proteins of a series of VVD mutants and the N- or C-terminal fragment of split firefly luciferase (NflucVVD52X and VVD52X-Cfluc). We then transfected a series of paired constructs in COS-7 cells and measured the bioluminescence from complemented split luciferase fragments (Figure 2a and Supplementary Figure 1). Mutations of Ile52 to amino acids with charged side chains (I52R, I52K, I52E, and I52D) completely disrupted homodimer formation, presumably because of electrostatic repulsion. In contrast, I52V, I52F, I52A, I52M, I52L, and I52W mutants that have hydrophobic side chains exhibited homodimer formation at a level comparable to that of the wild type. Among the I52X mutants examined, we found that only the I52C mutant (VVD-52C) exhibited substantially enhanced homodimerization activity, up to 180% of that of the wild type. Although the homodimerization activity at a dark condition was also enhanced up to 300%, VVD-52C has sufficient 13-fold induction. We investigated the effect of light irradiance in this assay and confirmed that there was no difference in light irradiance dependency between VVD and VVD-52C (Supplementary Figure 2). This indicates that the high homodimerization activity of VVD-52C does not arise from increased quantum yield of activation. Also, 30 W/m2 of light irradiation is enough to fully activate VVD and VVD-52C in this assay. 618

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Figure 2. Engineering the dimer interface of VVD. (a) Dimer-forming efficiency of I52X mutants. (b) Dimer-forming efficiency of VVD and VVD52C in the absence (left panel) or presence (right panel) of 10 mM DTT. (c) Dissociation kinetics of VVD-85V homodimer and that of VVD52C85V homodimer. (d) Western blot analysis for Nfluc-VVD-52C and VVD-52C-Cfluc. Error bars represent the SEM from (a) three individual experiments (n = 9), (b and c) two individual experiments (n = 6). Student’s two-tailed t test was performed. ***p < 0.001 versus wild type.

bioluminescence biosensor,24 we performed cell viability assays of HEK293 cells separately expressing paCasp9-WT, paCasp952C, iCasp9, and VVD (Figure 4b). We found that photoactivation of paCasp9-52C and AP20187-induced activation of iCasp9 could decrease luciferase activity by 52% and 93%, respectively. In contrast, HEK293 cells expressing paCasp9-WT did not show significant decrease of bioluminescence upon blue light irradiation. By fluorescence microscopic imaging, we confirmed HEK293 cells expressing paCasp9-WT did not show apoptosis morphology under blue light irradiation (Figure 4c). In contrast, cells expressing paCasp9-52C are degraded into cell fragments called apoptotic bodies after blue light irradiation, as with AP20187-treated cells expressing iCasp9 (Figure 4c).25 paCasp9-52C thus allows optical control of apoptosis. The results demonstrate that VVD-52C, but not wild-type VVD, has the potential to convert a chemically inducible tool into a photoactivatable tool. In conclusion, we have successfully improved dimer-forming efficiency of VVD up to 180% by introducing I52C mutation of its homodimer interface. Because I52C mutation also increases the dark state activity up to 300%, therefore the fold activation of VVD-52C (13-fold) is lower than that of wild-type (21-fold). This dark state activity of VVD-52C remains to be improved. Recently, rational site-directed mutagenesis that stabilizes the dark state structure of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) was reported to improve AsLOV2-

promoter having promoter activity weaker than that of SV40. Consequently, pΔSV40-GAVP showed little photoinduced reporter expression, presumably because the expression level of GAVP is too low to efficiently induce homodimerization of the transactivator. In contrast, pΔSV40-GAVP-52C still showed remarkable light-induced reporter expression with significant contrast despite its extremely low expression level (Figure 3c,d). The results indicate that GAVP-52C equipped with the present high affinity variant VVD-52C can more strongly induce reporter gene expression compared with GAVP having wild-type VVD. To demonstrate another utility of VVD-52C, we tried to convert a chemically inducible tool into a photoinducible tool using VVD-52C. As an example of chemically inducible tools, we focused on inducible caspase-9 (iCasp9), a chimeric protein of an FKBP mutant (FKBP36V) and catalytic domain of caspase-9 that is responsible for apoptosis.22,23 The small molecule AP20187 can induce homodimerization of FKBP36V and thereby dimerizes the catalytic domain of caspase-9. This increases the caspase-9 activity. Here we replace FKBP36V in iCasp9 with VVD-52C and develop photoactivatable caspase-9 (Figure 4a). We generated a fusion protein of VVD-52C and the catalytic domain of caspase-9 (paCasp9-52C). We also generated paCasp9-WT having wild-type VVD instead of VVD-52C. Using firefly luciferase as an adenosine triphosphate (ATP) 619

dx.doi.org/10.1021/cb400836k | ACS Chem. Biol. 2014, 9, 617−621

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Figure 3. Enhancing light-induced gene expression by VVD-52C. (a) Constructs for the two transactivators, GAVP and GAVP-52C. (b) Schematics of the gene expression system based on GAVP-52C. (c) Light-induced luciferase expression by GAVP (WT) and GAVP-52C (52C). SV40 and ΔSV40 promoters, respectively, are used for expression of the transactivators. Numbers in bar graph show the fold enhanced by using VVD-52C. (d) Fold induction of luciferase expression obtained in panel c. Error bars represent the SEM from three individual experiments (n = 9). Student’s two-tailed t test was performed. *p < 0.05. **p < 0.01.

Figure 4. Photoactivatable caspase-9. (a) Scheme of photoactivatable caspase-9 having VVD-52C (paCasp9-52C). (b) Photoinduced apoptosis by paCasp9-WT and paCasp9-52C. iCasp9 was used for AP20187-induced apoptosis. Cell death was evaluated by luciferasebased viability assay. (c) Apoptosis of HEK293 cells expressing each caspase-9 probe and mCherry. Allows indicate apoptotic bodies. Scale bar: 20 μm. Error bars represent the SEM from three independent experiments (n = 9). Student’s two-tailed t test was performed. ***p < 0.001.

based optogenetic tools.26 Similar efforts might increase the fold activation of VVD-52C. To demonstrate the competence of VVD-52C, we applied VVD-52C to the existing gene expression system based on VVD. In this system, known as the LightOn system, the lightinducible transactivator equipped with VVD-52C more strongly induced gene expression than that equipped with wild-type VVD. Furthermore, even when its expression was very low, the optogenetic tool equipped with VVD-52C drove signal transduction potently. This capacity of VVD-52C will be useful when a chimeric protein to be photoactivated by tagging with VVD-52C has cytotoxicity. The capacity of VVD-52C will also be useful for generating stable cell lines or transgenic organisms expressing VVD-52C-based optogenetic tools because these approaches often accompany low expression of exogenous genes. As exemplified with the development of photoactivatable caspase-9, we showed that VVD-52C has the potential to convert existing chemically induced homodimerzation tools to photoinducible tools. In contrast, replacing FKBP36V in chemically activatable iCasp9 with wild-type VVD completely removed induction potency because of low dimer-forming efficiency. In the past few decades, synthetic biologists have been creating various types of synthetic biology toolkits.3,14,27,28 Many chemically activatable tools that can drive cellular signaling based on homodimerization, such as caspase cascade,23,29,30 Fas-signaling,31 the mitogen activated protein kinase pathway,32 and receptor tyrosine kinase signaling,33 have been developed and used as both investigative and therapeutic tools. The present conversion approach using VVD-52C will make chemically activatable tools reborn as photoactivatable

tools, which can control cellular function with higher spatiotemporal resolution. We also have found that the present dimer-enhancing mutation (I52C) in the N-terminal helix of VVD is compatible with the I85V mutation in the cofactor-binding domain, which makes the dissociation kinetics of the homodimer much faster. The VVD-52C85V mutant with both enhanced dimer-forming efficiency and fast dissociation kinetics has the potential to more intensely switch on cellular signaling processes and more rapidly switch them off compared to wild-type VVD. In addition to fast mutants, represented by VVD-85V, other photocycle mutations in the cofactor-binding domain showing slow dissociation kinetics have also been explored.20 The present I52C mutation may enhance the dimer-forming efficiency of the beneficial mutants of VVD having a variety of dissociation kinetics and allow efficient photoregulation of cellular signaling processes on a broad range of time scales. VVD-52C, the present engineered photoinducible homodimerization system, will contribute to expanding the variety and usability of optogenetic toolkits.

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METHODS

For details see Supporting Information. 620

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(17) Clackson, T., Yang, W., Rozamus, L. W., Hatada, M., Amara, J. F., Rollins, C. T., Stevenson, L. F., Magari, S. R., Wood, S. A., Courage, N. L., Lu, X., Cerasoli, F., Gilman, M., and Holt, D. A. (1998) Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. U.S.A. 95, 10437−10442. (18) Vaidya, A. T., Chen, C. H., Dunlap, J. C., Loros, J. J., and Crane, B. R. (2011) Structure of a light-activated LOV protein dimer that regulates transcription. Sci. Signaling 4, ra50. (19) Paulmurugan, R., and Gambhir, S. S. (2007) Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying proteinprotein interactions. Anal. Chem. 79, 2346−2353. (20) Zoltowski, B. D., Vaccaro, B., and Crane, B. R. (2009) Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 5, 827−834. (21) Imayoshi, I., Isomura, A., Harima, Y., Kori, H., Miyachi, H., Fujiwara, T., Ishidate, F., and Kageyama, R. (2013) Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. Science 342, 1203−1208. (22) Straathof, K. C., Pulè, M. A., Yotnda, P., Dotti, G., Vanin, E. F., Brenner, M. K., Heslop, H. E., Spencer, D. M., and Rooney, C. M. (2005) An inducible caspase 9 safety switch for T-cell therapy. Blood 105, 4247−4254. (23) Di Stasi, A., Tey, S.-K., Dotti, G., Fujita, Y., Kennedy-Nasser, A., Martinez, C., Straathof, K., Liu, E., Durett, A. G., Grilley, B., Liu, H., Cruz, C. R., Savoldo, B., Gee, A. P., Schindler, J., Krance, R. a, Heslop, H. E., Spencer, D. M., Rooney, C. M., and Brenner, M. K. (2011) Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 365, 1673−1683. (24) Crouch, S. P., Kozlowski, R., Slater, K. J., and Fletcher, J. (1993) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Methods 160, 81−88. (25) Van Cruchten, S., and Van Den Broeck, W. (2002) Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat. Histol. Embryol. 31, 214−223. (26) Strickland, D., Yao, X., Gawlak, G., Rosen, M. K., Gardner, K. H., and Sosnick, T. R. (2010) Rationally improving LOV domainbased photoswitches. Nat. Methods 7, 623−626. (27) DeRose, R., Miyamoto, T., and Inoue, T. (2013) Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Arch. 465, 409−417. (28) Aubel, D., and Fussenegger, M. (2010) Mammalian synthetic biology-from tools to therapies. Bioessays 32, 332−345. (29) Fan, L., An, K. W. F., Khan, T., Pham, E., and Spencer, D. M. (1999) Improved artificial death switches based on caspases and FADD. Hum. Gene Ther. 10, 2273−2285. (30) MacCorkle, R. A, Freeman, K. W., and Spencer, D. M. (1998) Synthetic activation of caspases: artificial death switches. Proc. Natl. Acad. Sci. U.S.A. 95, 3655−3660. (31) Spencer, D. M., Belshaw, P. J., Chen, L., Ho, S. N., Randazzo, F., Crabtree, G. R., and Schreiber, S. L. (1996) Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization. Curr. Biol. 6, 839−847. (32) Cheng, J., Yu, L., Zhang, D., Huang, Q., Spencer, D., and Su, B. (2005) Dimerization through the catalytic domain is essential for MEKK2 activation. J. Biol. Chem. 280, 13477−13482. (33) Muthuswamy, S. K., Li, D., Lelievre, S., Bissell, M. J., and Brugge, J. S. (2001) ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nat. Cell Biol. 3, 785− 792.

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), Japan Society for the Promotion of Science (JSPS), and Research Foundation for Opto-Science and Technology (to M.S.).

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REFERENCES

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