Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence

Hisatomi, Osamu; Nakatani, Yôichi; Takeuchi, Ken; Takahashi, Fumio; Kataoka, Hironao

doi:10.1074/jbc.m114.554618

Cited by 44 publications

(135 citation statements)

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“…The half-life times (τ 1/2 ) of the reactions were calculated at 7.1−7.6 min, which were comparable with that for the ZL proteins (τ 1/2 = 7.2 ± 0.4 min). 28,29 Taken together, these results suggest that the N-terminal truncation of PZ had negligible effects on the π-electron state and photoreactivity of the LOV domain in the recombinant proteins. Table 1.…”

Section: Construction Of Expressionmentioning

confidence: 79%

Molecular Mechanism of Photozipper, a Light-Regulated Dimerizing Module Consisting of the bZIP and LOV Domains of Aureochrome-1

Nakatani

Hisatomi

2015

Biochemistry

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Aureochrome-1 (AUREO1) is a blue light (BL) receptor responsible for the BL-induced blanching of a stramenopile alga, Vaucheria frigida. The AUREO1 protein contains a central basic region/leucine zipper (bZIP) domain, and a C-terminal light-oxygen-voltage-sensing (LOV) domain. BL induces the dimerization of monomeric AUREO1, which subsequently increases the affinity of this transcription factor for its target DNA [Hisatomi, O., et al. (2014) J. Biol. Chem. 289, 17379-17391]. We constructed a synthetic gene encoding N-terminally truncated monomeric AUREO1 (designated Photozipper) to elucidate the molecular mechanism of this BL-regulated transcription factor and to develop it as an optogenetic tool. In this study, four different Photozipper (PZ) protein constructs were prepared comprising different N-terminal truncations. The monomer-dimer equilibria of the PZ constructs were investigated in the dark and light states. Dynamic light scattering and size-exclusion chromatography analyses revealed that the apparent dissociation constants of PZ dimers with and without the ZIP region were ~100 and 30 μM, respectively, indicating that the ZIP region stabilized the monomeric form in the dark state. In the light state, fluorescence resonance energy transfer analyses demonstrated that deletion of the ZIP region increased the dissociation constant from ~0.15 to 0.6 μM, suggesting that intermolecular LOV-LOV and ZIP-ZIP interactions stabilized the dimeric forms. Our results suggest that synergistic interactions between the LOV and bZIP domains stabilize the monomeric form in the dark state and the dimeric form in the light state, which possibly contributes to the function of PZ as a BL-regulated molecular switch.

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Section: Construction Of Expressionmentioning

confidence: 79%

Molecular Mechanism of Photozipper, a Light-Regulated Dimerizing Module Consisting of the bZIP and LOV Domains of Aureochrome-1

Nakatani

Hisatomi

2015

Biochemistry

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“…Existing photochemical and structural analyses show that, by and large, these structures do not form stable dimers in the dark [bZIPs such as aureochrome (63), zinc fingers such as White Collar (64)(65)(66), HTH proteins such as EL222 (67), and short LOVs such as VIVID (7,(37)(38)(39)] or are oriented in antiparallel fashion inconsistent with a parallel extended coiled-coil model, such as phototropins (68) and F-box/ Kelch repeats like FKF1 (69,70). Thus, the observed trend of linker length discretization by effector type and phylum of origin (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functional and topological diversity of LOV domain photoreceptors

Glantz

Carpenter

Melkonian

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.photoreceptors | LOV | flavoproteins | optogenetics

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“…Where E 0 = -352 mV at pH 7.4, R is the gas constant, T is the absolute temperature, and F is Faraday's constant, n = 2, and [DTT oxi ] and [DTT red ] are molar concentrations of oxidized and reduced DTT, respectively (36). Subsequently, recombinant wild-type CBS (100 μg) was preincubated in solutions (500 μl) with various concentrations of DTT oxi and DTT red for 1 h at 37 °C, and then precipitated with 15% (v/v) trichloroacetic acid at 4 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bond

Niu¹,

Wang²,

Qian

et al. 2018

Journal of Biological Chemistry

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Cystathionine β-synthase (CBS) is the central enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of the three major enzymes involved in the biogenesis of H 2 S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a C 272 XXC 275 motif whose function has yet to be determined. In the present study, we demonstrated that the CXXC motif exists in oxidized and reduced states in the recombinant enzyme by mass spectroscopic analysis and a thiol labeling assay. The activity of reduced CBS is ~2-to 3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys 272 -Cys 275 disulfide bond in CBS has a midpoint potential of -314 mV at pH 7.4. Additionally, the CXXC motif also exists in oxidized and reduced states in human embryonic kidney 293 (HEK293) cells under oxidative and reductive conditions, and stressing these cells with dithiothreitol (DTT) results in more reduced enzyme and a concomitant increase in H 2 S production in live HEK293 cells as determined using a H 2 S fluorescent probe. By contrast, incubation of cells with aminooxyacetic acid (AOAA), an inhibitor of CBS and cystathionine γ-lyase (CSE), eliminates the increase of H 2 S production after the cells were exposed to DTT. These findings indicate that CBS is post-translationally regulated by a redox-active disulfide bond in the CXXC motif. The results als o demonstrate that CBS-derived H 2 S production is increased in cells under reductive stress conditions.

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Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence

Cited by 44 publications

References 39 publications

Molecular Mechanism of Photozipper, a Light-Regulated Dimerizing Module Consisting of the bZIP and LOV Domains of Aureochrome-1

Molecular Mechanism of Photozipper, a Light-Regulated Dimerizing Module Consisting of the bZIP and LOV Domains of Aureochrome-1

Functional and topological diversity of LOV domain photoreceptors

Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bond

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