Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 Å and 2.0 Å, respectively. Either LOV1 domain forms a dimer through face-to-face association of β-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their β-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the β-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.     

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.     

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.     

Higher plants use LOV to perceive blue light

Higher plants use several classes of blue light receptors to modulate a wide variety of physiological responses. Among them, both the phototropins and members of the Zeitlupe (ZTL) family use light oxygen voltage (LOV) photosensory domains. In Arabidopsis, these families comprise phot1, phot2 and ZTL, LOV Kelch Protein 2 (LKP2), and Flavin-binding Kelch F-box1 (FKF1). It has now been convincingly shown that blue-light-induced autophosphorylation of the phot1 kinase domain is an essential step in signal transduction. Recent experiments also shed light on the partially distinct photosensory specificities of phot1 and phot2. Phototropin signaling branches rapidly following photoreceptor activation to mediate distinct responses such as chloroplast movements or phototropism. Light activation of the LOV domain in ZTL family members modulates their capacity to interact with GIGANTEA (GI) and their ubiquitin E3 ligase activity. A complex between GI and FKF1 is required to trigger the degradation of a repressor of CO (CONSTANS) expression and thus modulates flowering time. In contrast, light-regulated complex formation between ZTL and GI appears to limit the capacity of ZTL to degrade its targets, which are part of the circadian oscillator.     

LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1

Phototropin is a blue light receptor in plants and is thought to be a light-regulated protein kinase. Previously, we defined the role of the photoreceptive domains, LOV1 and 2, in the light activation of the kinase in Arabidopsis phototropin2 (phot2). In this study, photoregulation of the kinase in phototropin1 (phot1) was studied using LOV2-linker-kinase polypeptide. We designed a new substrate consisting of the N-terminal part of the phot1 with autophosphorylation sites. The LOV2-linker-kinase had the same spectroscopic properties as those of the LOV2 core and phosphorylated the substrate in a light-dependent manner. Amino acid substitution experiments proved that the phosphorylation comes from the activation of the kinase via photoreaction of LOV2.     

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate,S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin,a Plant Blue Light Receptor

Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391–3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.     

Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.     

Phototropin receptor kinase activation by blue light

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central β-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation.Key words: phototropin, LOV domain, FMN, cysteinyl adduct, amphipathic helix, receptor autophosphoryation     

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.     

Mapping of low- and high-fluence autophosphorylation sites in phototropin 1

Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins.     

Light Signal Transduction Pathway from Flavin Chromophore to the Jα Helix of Arabidopsis Phototropin1

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jα helix) for the function of phototropin; however, it remains unclear how the Jα helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jα) of the Jα helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (−)/1625 (+) cm⁻¹) of LOV2-Jα at physiological temperatures (≥260 K), corresponding to structural perturbation of the α-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the β-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jα helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jα helix in Arabidopsis phot1.     

Stability of dimer and domain-domain interaction of Arabidopsis phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.     

A LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.     

Subcellular localization and turnover of Arabidopsis phototropin 1

We reported recently that internalization of the plant blue light receptor phototropin 1 (phot1) from the plasma membrane in response to irradiation is reliant on receptor autophosphorylation. Pharmacological interference and co-immunoprecipitation analyses also indicated that light-induced internalization of phot1 involves clathrin-dependent processes. Here, we describe additional pharmacological studies that impact the subcellular localization and trafficking of Arabidopsis phot1. Alterations in the microtububle cytoskeleton led to dramatic differences in phot1 localization and function. Likewise, inhibition of phosphatidic acid (PA) signaling was found to impair phot1 localization and function. However, action of PA inhibition on phot1 function may be attributed to pleiotropic effects on cell growth. While phot1 kinase activation is necessary to stimulate its internalization, autophosphorylation is not required for phot1 turnover in response to prolonged blue light irradiation. The implications of these findings in regard to phot1 localization and function are discussed.Key words: phototropin 1 (phot1), phototropism, subcellular trafficking, autophosphorylation, protein turnover     

The Arabidopsis rcn1-1 Mutation Impairs Dephosphorylation of Phot2, Resulting in Enhanced Blue Light Responses

Phototropins (phot) sense blue light through the two N-terminal chromophore binding LOV domains and activate the C-terminal kinase domain. The resulting phototropin autophosphorylation is essential for biological activity. We identified the A1 subunit of Ser/Thr protein phosphatase 2A (PP2A) as interacting with full-length phot2 in yeast and also interacting with phot2 in an in vitro protein binding assay. Phenotypic characterizations of a phot1-5 rcn1-1 (for root curling in n-naphthylphthalamic acid1) double mutant, in which phot2 is the only functional phototropin and PP2A activity is reduced, showed enhanced phototropic sensitivity and enhanced blue light–induced stomatal opening, suggesting that PP2A activity is involved in regulating phot2 function. When treated with cantharidin, a chemical inhibitor of PP2A, the phot1-5 mutant exhibited enhanced phot2-mediated phototropic responses like those of the phot1-5 rcn1-1 double mutant. Immunoblot analysis to examine phot2 endogenous phosphorylation levels and in vitro phosphorylation assays of phot2 extracted from plants during dark recovery from blue light exposure confirmed that phot2 is more slowly dephosphorylated in the reduced PP2A activity background than in the wild-type PP2A background, suggesting that phosphorylated phot2 is a substrate of PP2A activity. While reduced PP2A activity enhanced the activity of phot2, it did not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism or stomatal opening.     

Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot

Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.     

Light-induced movement of the LOV2 domain in an Asp720Asn mutant LOV2-kinase fragment of Arabidopsis phototropin 2

Phototropin, a blue-light receptor protein of plants, triggers phototropic responses, chloroplast relocation, and opening of stomata to maximize the efficiency of photosynthesis. Phototropin is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) that absorb blue light and a serine/theroine kinase domain responsible for light-dependent autophosphorylation leading to cellular signaling cascades. Although the light-activated LOV2 domain is primarily responsible for subsequent activation of the kinase domain, it is unclear how conformational changes in the former transmit to the latter. To understand this molecular mechanism in Arabidopsis phototropin 2, we performed small-angle X-ray scattering analysis on a fragment composed of the LOV2 and kinase domains, which contained an Asp720Asn mutation that led to an absence of ATP binding activity. The scattering data were collected up to a resolution of 25 ?. The apparent molecular weight of the fragment estimated from scattering intensities demonstrated that the fragment existed in a monomeric form in solution. The fragment exhibited photoreversible changes in the scattering profiles, and the radii of gyration under dark and blue-light irradiation conditions were 32.4 and 34.8 ?, respectively. In the dark, the molecular shape restored from the scattering profile appeared as an elongated shape of 110 ? in length and 45 ? in width. The homology modeled LOV2 and kinase domains could be fitted to the molecular shape and appeared to make slight contact. However, under blue-light irradiation, a more extended molecular shape was observed. The changes in the molecular shape and radius of gyration were interpreted as a light-dependent positional shift of the LOV2 domain of approximately 13 ? from the kinase domain. Because the region connecting the LOV2 and kinase domains was categorized as a naturally unfolded polypeptide, we propose that the light-activated LOV2 domain triggers conformational changes in the linker region to separate the LOV2 and kinase domains.     

Oligomeric structure of LOV domains in Arabidopsis phototropin

Oligomeric structures of the four LOV domains in Arabidopsis phototropin1 (phot1) and 2 (phot2) were studied using crosslinking. Both LOV1 domains of phot1 and phot2 form a dimer independently on the light conditions, suggesting that the LOV1 domain can be a stable dimerization site of phot in vivo. In contrast, phot1-LOV2 is in a monomer-dimer equilibrium and phot2-LOV2 exists as a monomer in the dark. Blue light-induced a slight increase in the monomer population in phot1-LOV2, suggesting a possible blue light-inducible dissociation of dimers. Furthermore, blue light caused a band shift of the phot2-LOV2 monomer. CD spectra revealed the unfolding of helices and the formation of strand structures. Both light-induced changes were reversible in the dark.

Structured summary

MINT-6823377, MINT-6823391:PHOT1 (uniprotkb:O48963) and PHOT1 (uniprotkb: O48963) bind (MI:0407) by cross-linking studies (MI:0030)MINT-6823495, MINT-6823508:PHOT2 (uniprotkb:P93025) and PHOT2 (uniprotkb:P93025) bind (MI:0407) by cross-linking studies (MI:0030)