Satoru Tokutomi

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 A and 2.0 A, respectively. Either LOV1 domain forms a dimer through face-to-face association of beta-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their beta-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the beta-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.

Ca2+-induced phase separation in phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca2+-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidylethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94--100% at 23 degrees C and 84--88% at 40 degrees C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74--85% at 23 degrees C and 61--79% at 40 degrees C. Ca2+ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was 1.4 . 10(-7) M for phosphatidylserine-phosphatidylethanolamine and 1.2 . 10(-6) M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca2+ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17 degrees C) and phosphatidylcholine (-15 degrees C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca2+-chelated solid-phase phosphatidylserine domain.

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.

Oligomeric structure of LOV domains in Arabidopsis phototropin

PHOT2 (uniprotkb:P93025) and PHOT2 (uniprotkb:P93025) bind (MI:0407) by cross‐linking studies (MI:0030)

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.

Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners

The light, oxygen, or voltage (LOV) domain that belongs to the Per-ARNT-Sim (PAS) domain superfamily is a blue light sensory module. The Arabidopsis thalianaPAS/LOV PROTEIN (PLP) gene encodes three putative blue light receptor proteins, PLPA, PLPB, and PLPC, because of its mRNA splicing variation. PLPA and PLPB each contain one PAS domain at the N-terminal region and one LOV domain at the C-terminal region, while the LOV domain is truncated in PLPC. RNA gel blot analysis showed that PLP mRNA was markedly expressed after exposure to salt or dehydration stress. Yeast two-hybrid screening led to the isolation of VITAMIN C DEFECTIVE 2 (VTC2), VTC2-LIKE (VTC2L), and BEL1-LIKE HOMEODOMAIN 10 proteins (BLH10A and BLH10B) as PLP-interacting proteins. The molecular interaction of PLPA with VTC2L, BLH10A or BLH10B, and that of PLPB with VTC2L were diminished when yeasts were grown under blue light illumination. Furthermore, the possible binding of flavin chromophore to PLPA and PLPB was demonstrated. These results imply that the LOV domain of PLPA and PLPB functions as a blue light sensor, and suggest the applicability of these interactions to blue light-dependent switching in transcriptional regulation in yeast or other organisms.

Involvement of Electron Transfer in the Photoreaction of Zebrafish Cryptochrome‐DASH†

Photoreaction of a blue‐light photoreceptor Cryptochrome‐DASH (Cry‐DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV–visible absorption spectroscopy in aqueous solutions at 293u2003K. Zebrafish Cry‐DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10‐methenyl‐5,6,7,8‐tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FADox) in the dark. Blue light converts FADox to the neutral radical form (FADH?). Formed FADH? is transformed to the fully reduced form FADH2 (or FADH−) by successive light irradiation, or reverts to FADox. FADH2 (or FADH−) reverts to FADH? or possibly to FADox directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry‐DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry‐DASH family protein.

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.

Vibrational assignment of the flavin-cysteinyl adduct in a signaling state of the LOV domain in FKF1.

LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins in organism ranging from archaea to eukaryotes, and they noncovalently bind a single flavin mononucleotide as a chromophore. We report the Raman spectra of the dark state of LOV domain in FKF1 from Arabidopsis thaliana. Spectra have been also measured for the signaling state, where a cysteinyl-flavin adduct is formed upon light irradiation. Most of the observed Raman bands are assigned on the basis of normal mode calculations using a density functional theory. We also discuss implication for the analysis of the infrared spectra of LOV domains. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in LOV domains by vibrational spectroscopy.

PAS/LOV proteins: A proposed new class of plant blue light receptor

The light, oxygen, or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato, and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor. Addendum to: Ogura Y, Komatsu A, Zikihara K, Nanjo T, Tokutomi S, Wada M, Kiyosue T. Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners. J Plant Res 2008; 121: 97–105.