Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome

Thöing, Christian; Oldemeyer, Sabine; Kottke, Tilman

doi:10.1021/jacs.5b01404

Cited by 54 publications

(103 citation statements)

References 59 publications

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“…B, double difference spectrum of the 1 s spectrum minus the steady state spectrum was calculated. The comparison to reference spectra of TyrO ⅐ in water (red) (29) and at 140 K (blue) (30) reveals a red shift. A complete match is obtained by shifting the reference spectrum by 9 nm (inset).…”

Section: Resultsmentioning

confidence: 99%

“…2B). The comparison to reference spectra (29,30) revealed that such a band is characteristic for TyrO ⅐ . In contrast to the reference spectra obtained in water, TyrO ⅐ in aCRY is red-shifted by the protein environment by 9 nm (inset, Fig.…”

Section: Long-lived Tyrosyl Radical In the Animal-like Cryptochromementioning

confidence: 90%

“…The experimental setup for time-resolved UV-vis spectroscopy on slowly recovering systems has been described previously (29). aCRY was pre-illuminated for 15 s under stirring to generate FADH ⅐ using a 455 nm LED (Luxeon Star, Lumileds) with an intensity of 10 milliwatt/cm 2 at the sample (FWHM of 20 nm).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, time-resolved UV-visible (UV-vis) spectroscopy on cryptochromes and photolyases has revealed the involvement of transient radical species formed from FAD, tryptophan, and tyrosine as part of the electron transfer cascade (21)(22)(23)(24)(25)(26)(27). Tryptophan neutral radicals (Trp ⅐ ) exhibit a broad band centered at around 510 nm (28), whereas tyrosyl radicals (TyrO ⅐ ) are characterized by two sharp, adjacent bands at 388 and 408 nm (29,30). Moreover, the formation of these amino acid radicals in the electron transfer cascade has been disclosed by time-resolved EPR studies (31,32).…”

Section: * This Work Was Supported By the Deutsche Forschungsgemeinscmentioning

confidence: 99%

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Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

Oldemeyer

Franz

Wenzel

et al. 2016

Journal of Biological Chemistry

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Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii. This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UVvisible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 s after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6 -4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mM injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner.Cryptochromes represent a group of diverse sensory photoreceptors present in all kingdoms of life (1, 2). Together with the UV-light-dependent DNA repair enzymes, the photolyases (3), they constitute the cryptochrome/photolyase family. Members of this family share a highly conserved photolyase homology region (PHR) 2 , which comprises ϳ500 amino acids and carries a non-covalently bound flavin adenine dinucleotide (FAD) as a chromophore. The C-terminal extension (CCT) present in many cryptochromes and photolyases is strongly variable in amino acid composition and length and has been shown to be crucial for signal transduction in the Arabidopsis cryptochrome AtCRY1 (4). The diverse subfamilies of cryptochromes comprise proteins acting as central blue light sensors in bacteria, fungi, plants, and insects (animal type I CRY) (1). Moreover, CRYs are also found as the light-independent, central part of the oscillator of the biological clock in mammals (animal type II CRY) (5) and as a mediator for light-dependent magnetosensitivity in flies (6). Opposed to these findings, DASH cryptochromes have been found to repair lesions in single-stranded DNA and double-stranded loop-structured DNA in vitro (7,8). Therefore, DASH cryptochromes are more similar in terms of functionality to photolyases than cryptochromes. Among the photolyases, two different types are separated dependin...

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Section: Resultsmentioning

confidence: 99%

Section: Long-lived Tyrosyl Radical In the Animal-like Cryptochromementioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: * This Work Was Supported By the Deutsche Forschungsgemeinscmentioning

confidence: 99%

See 2 more Smart Citations

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

Oldemeyer

Franz

Wenzel

et al. 2016

Journal of Biological Chemistry

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“…It has been previously shown that conformational change involving the cry1 C-terminal domain occurs on a millisecond time scale, 33 which is too slow for triggering by direct electron and/or proton transfer events. More recent data suggests conformational change may be initiated already within the N-terminal domain at much more rapid time scales, 34 consistent with triggers such as deprotonation of D396, an aspartic acid residue in proximity to the flavin.…”

Section: The C-terminal Domain Of Cryptochrome Is Implicated In Strucmentioning

confidence: 54%

Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1

El‐Esawi

Glascoe

Engle

et al. 2015

Plant Signaling & Behavior

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Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the 'Trp triad.' Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH ) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.

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Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome

Wen

Shao

et al. 2019

ChemBioChem

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Plant cryptochromes (CRYs) are blue‐light receptors that regulate light‐dependent growth, development, and circadian rhythms. A flavin adenine dinucleotide (FAD) cofactor is bound to the photolyase homology region (PHR) of plant CRYs and can be photoreduced to a neutral radical state under blue light. This photoreaction can trigger subsequent signal transduction. Plant CRYs can also bind an ATP molecule adjacent to FAD in a pocket of the PHR. Chlamydomonas reinhardtii contains a single plant CRY, named Chlamydomonas photolyase homologue 1 (CPH1). In CPH1, Cys392 and Asp393 are located near the FAD cofactor. Here we have shown that replacing Cys392 with Ser has little effect on the properties of CPH1. The C392N mutant, however, showed a faster photoreduction rate than wild‐type CPH1, together with a significantly lower oxidation rate of the neutral radical state. Substituting an Asn residue for Asp393 in CPH1 improved the binding affinity for FAD as well as the stability of the neutral radical, but photoreduction in the case of this mutant was severely inhibited. In the presence of ATP, CPH1 and its mutants exhibited significantly higher binding affinity for FAD and slower oxidation of the neutral radical. These results reveal that the residues at site 392 and the presence of ATP can tune the stability of the neutral radical, that the Asp residue at site 393 is crucial for photoreduction, and that the photoreduction rate is not determined merely by the stability of the neutral radical in CPH1.

show abstract

Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome

Cited by 54 publications

References 59 publications

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1

Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome

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