Abstract
Phototropin-like LOV domains form a cysteinyl-flavin adduct in response to blue light but show considerable variation in output signal and the lifetime of the photo-adduct signaling state. Mechanistic studies of the slow-cycling fungal LOV photoreceptor Vivid (VVD) reveal the importance of reactive cysteine conformation, flavin electronic environment and solvent accessibility for adduct scission and thermal reversion. Proton inventory, pH effects, base catalysis and structural studies implicate flavin N5 deprotonation as rate-determining for recovery. Substitutions of active site residues Ile74, Ile85, Met135 and Met165 alter photoadduct lifetimes by over four orders of magnitude in VVD, and similar changes in other LOV proteins show analogous effects. Adduct state decay rates also correlate with changes in conformational and oligomeric properties of the protein necessary for signaling. These findings link natural sequence variation of LOV domains to function and provide a means to design broadly reactive light-sensitive probes.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Crosson, S., Rajagopal, S. & Moffat, K. The LOV domain family: photoresponsive signaling modules coupled to diverse output domains. Biochemistry 42, 2–10 (2003).
Christie, J.M. Phototropin blue-light receptors. Annu. Rev. Plant Biol. 58, 21–45 (2007).
Losi, A., Polverini, E., Quest, B. & Gartner, W. First evidence for phototropin-related blue-light receptors in prokaryotes. Biophys. J. 82, 2627–2634 (2002).
Purcell, E.B., Siegal-Gaskins, D., Rawling, D.C., Fiebig, A. & Crosson, S. A photosensory two-component system regulates bacterial cell attachment. Proc. Natl. Acad. Sci. USA 104, 18241–18246 (2007).
Schmoll, M., Franchi, L., Kubicek, C.P. & Envoy, A. PAS/LOV domain protein of Hypocrea jecorina (anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light. Eukaryot. Cell 4, 1998–2007 (2005).
Schwerdtfeger, C. & Linden, H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J. 22, 4846–4855 (2003).
Swartz, T.E. et al. Blue-light-activated histidine kinases: two component sensors in bacteria. Science 317, 1090–1093 (2007).
Zoltowski, B.D. et al. Conformational switching in the fungal light sensor vivid. Science 316, 1054–1057 (2007).
Froehlich, A., Liu, Y., Loros, J.J. & Dunlap, J.C. White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 297, 815–819 (2002).
Moglich, A. & Moffat, K. Structural basis for light-dependent signalling in the dimeric LOV domain of the photosensor YtvA. J. Mol. Biol. 373, 112–126 (2007).
Cao, Z., Buttani, V., Losi, A. & Gartner, W. A blue light inducible two-component signal transduction system in the plant pathogen Pseudomonas syringae pv. tomato. Biophys. J. 94, 897–905 (2008).
Zikihara, K. et al. Photoreaction cycle of the light, oxygen and voltage domain in FKF1 determined by low-temperature absorption spectroscopy. Biochemistry 45, 10828–10837 (2006).
Zoltowski, B.D. & Crane, B.R. Light activation of the LOV protein VVD generates a rapidly exchanging dimer. Biochemistry 47, 7012–7019 (2008).
Nakasako, M., Kazunori, Z., Matsuoka, D., Katsura, H. & Tokutomi, S. Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2. J. Mol. Biol. 381, 718–733 (2008).
Nakasone, Y. et al. Stability of dimer and domain-domain interaction of Arabidopsis phototropin 1 LOV2. J. Mol. Biol. 383, 904–913 (2008).
Corchnoy, S.B. et al. Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1. J. Biol. Chem. 278, 724–731 (2003).
Kennis, J.T.M. et al. Primary reactions of the LOV2 domain of photoropin, a plant blue-light photoreceptor. Biochemistry 42, 3385–3392 (2003).
Swartz, T.E. et al. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J. Biol. Chem. 276, 36493–36500 (2001).
Kottke, T., Heberle, J., Hehn, D., Dick, B. & Hegemann, P. Phot-LOV1: photocycle of a blue-light receptor domain from the green alga Chlamydomonas reinhardtii. Biophys. J. 84, 1192–1201 (2003).
Alexandre, M.T.A. et al. Mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain. Biochemistry 46, 3129–3137 (2007).
Christie, J.M. et al. Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1. Biochemistry 46, 9310–9319 (2007).
Elvin, M., Loros, J.J., Dunlap, J.C. & Heintzen, C. The PAS/LOV protein VVD supports a rapidly dampened daytime oscillator that facilitates entrainment of the Neurospora circadian clock. Genes Dev. 19, 2593–2605 (2005).
Heintzen, C., Loros, J.J., Dunlap, J.C. & The, P.A.S. Protein VIVID defines a Clock-associated feedback loop that represses light input, modulates gating, and regulates Clock resetting. Cell 104, 453–464 (2001).
Shrode, L.B., Lewis, Z.A., White, L.D., Bell-Pedersen, D. & Ebbole, D.J. vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation. Fungal Genet. Biol. 32, 169–181 (2001).
Losi, A., Quest, B. & Gartner, W. Listening to the blue: the time resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain. Photochem. Photobiol. Sci. 2, 759–766 (2003).
Kennis, J.T.M. et al. The LOV2 domain of phototropin: a reversible photochromic switch. J. Am. Chem. Soc. 126, 4512–4513 (2004).
Kasahara, M. et al. Photochemical properties of the flavin mononucleotide-binding domains of the photorropins from Arabidopsis, rice, and Chlamydomonas reinhardtii. Plant Physiol. 129, 762–773 (2002).
Druhan, L.J. & Swenson, R.P. Role of methionine 56 in the control of the oxidation-reduction potentials of the Clostridium beijerinckii flavodoxin: effects of substitutions by aliphatic amino acids and evidence for a role of sulfur-flavin interactions. Biochemistry 37, 9668–9678 (1998).
Alexandre, M.T., van Grondelle, R., Hellingwerf, K.J., Robert, B. & Kennis, J.T. Perturbation of the ground-state electronic structure of FMN by the conserved cysteine in phototropin LOV2 domains. Phys. Chem. Chem. Phys. 10, 6693–6702 (2008).
Venkatasubban, K.S. & Schowen, R.L. The proton inventory technique. CRC Crit. Rev. Biochem. 17, 1–44 (1984).
Fedorov, R. et al. Crystal structures and molecular mechanism of a light-induced signaling switch: the Phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys. J. 84, 2474–2482 (2003).
Sato, Y. et al. Heterogeneous environment of the S-H Group of Cys966 near the flavin chromophore in the LOV2 domain of Adiantum neochrome 1. Biochemistry 46, 10258–10265 (2007).
Lamb, J.S. et al. Illuminating solution responses of a LOV-domain protein with photocoupled small angle X-ray scattering. J. Mol. Biol. (in the press).
Yamamoto, A., Iwata, T., Tokutomi, S. & Kandori, H. Role of Phe1010 in light-induced structural changes of the neo1–LOV2 domain of Adiantum. Biochemistry 47, 922–928 (2008).
Ishikita, H. Influence of the protein environment on the redox potentials of flavodoxins form Clostridium beijerinckii. J. Biol. Chem. 282, 25240–25246 (2007).
Yagi, K., Ohishi, N., Nishimoto, K., Choi, J.D. & Song, P.S. Effect of hydrogen bonding on electronic spectra and reactivity of flavins. Biochemistry 19, 1553–1557 (1980).
Fedorov, R. et al. Crystal structures and molecular mechanism of a light-induced signaling switch: the Phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys. J. 84, 2474–2482 (2003).
Iwata, T. et al. Light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 studied by low-temperature FTIR and UV-visible spectroscopy. Biochemistry 42, 8183–8191 (2003).
Iwata, T., Tokutomi, S. & Kandori, H. Photoreaction of the cysteine S-H group in the LOV2 domain of Adiantum phytochrome3. J. Am. Chem. Soc. 124, 11840–11841 (2002).
Sato, Y., Iwata, T., Tokutomi, S. & Kandori, H. Reactive cysteine is protonated in the triplet excited state of the LOV2 domain in Adiantum phytochrome3. J. Am. Chem. Soc. 127, 1088–1089 (2005).
Schleicher, E. et al. On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin. J. Am. Chem. Soc. 126, 11067–11076 (2004).
Dittrich, M., Freddolino, P.L. & Schulten, K. When light falls in LOV: a quantum mechanical/molecular mechanical study of photoexcitation in Phot-LOV1 of Chlamydomonas reinhardtii. J. Phys. Chem. B 109, 13006–13013 (2005).
Domratcheva, T., Fedorov, R. & Schlichting, I. Analysis of the primary photocycle reactions occurring in the light, oxygen, and voltage blue-light receptor by multiconfigurational quantum-chemical methods. J. Chem. Theory Comput. 2, 1565–1574 (2006).
Nash, A.I., Ko, W.H., Harper, S.M. & Gardner, K.H. A conserved glutamine plays a central role in LOV domain signal transmission and its duration. Biochemistry 47, 13842–13849 (2008).
Yoshida, Y. & Hasunuma, K. Reactive oxygen species affect photomorphogenesis in Neurospora crassa. J. Biol. Chem. 279, 6986–6993 (2004).
Strickland, D., Moffat, K. & Sosnick, T.R. Light activated DNA binding in a designed allosteric protein. Proc. Natl. Acad. Sci. USA 105, 10709–10714 (2008).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).
McRee, D.E. XtalView: a visual protein crystallographic software system for X11/Xview. J. Mol. Graph. 10, 44–47 (1992).
Brunger, A.T. et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Acknowledgements
The authors thank A. Vaidya for help with kinetic studies, K. Gardner (University of Texas Southwestern) for supplying the AsLOV2 expression clone, J. Widom for help with mutagenesis and protein expression, and the Cornell High Energy Synchrotron for access to data collection facilities. This work was supported by US National Institutes of Health grant R01- GM079679.
Author information
Authors and Affiliations
Contributions
B.D.Z., B.V. and B.R.C. designed experiments; B.D.Z. and B.V. carried out experiments; B.D.Z., B.V. and B.R.C. analyzed data; B.D.Z. and B.R.C. wrote the manuscript.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 2382 kb)
Rights and permissions
About this article
Cite this article
Zoltowski, B., Vaccaro, B. & Crane, B. Mechanism-based tuning of a LOV domain photoreceptor. Nat Chem Biol 5, 827–834 (2009). https://doi.org/10.1038/nchembio.210
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.210
This article is cited by
-
Controlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction
Nature Communications (2023)
-
Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins
Nature Biotechnology (2022)
-
Synthetic cells with self-activating optogenetic proteins communicate with natural cells
Nature Communications (2022)
-
Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery
Photochemical & Photobiological Sciences (2022)
-
Engineering AraC to make it responsive to light instead of arabinose
Nature Chemical Biology (2021)