Oligomeric structure of LOV domains in <i>Arabidopsis</i> phototropin

Katsura, Hitomi; Zikihara, Kazunori; Okajima, Koji; Yoshihara, Shizue; Tokutomi, Satoru

doi:10.1016/j.febslet.2009.01.019

Cited by 35 publications

(35 citation statements)

References 23 publications

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“…This fact suggests that LOV1 domains of angiosperm LLPs may have lacked the cysteine during their evolution. The LOV1 domains of A. thaliana phototropin 1 and 2 are proposed to function as a dimerization site for the phototropins (26). Interestingly, the dimer formation is light-independent (26).…”

Section: Discussionmentioning

confidence: 99%

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains, LOV/LOV Proteins

Kasahara

Torii

Fujita

et al. 2010

Journal of Biological Chemistry

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Four photoreceptor families have been found from plants including phytochrome, cryptochrome, phototropin, and ADO/ FKF/LKP/ZTL family proteins. Among them, phototropin and ADO/FKF/LKP/ZTL use a common light-sensing domain, LOV domain (1). The LOV domain was found as the lightsensing domain of the phototropin (PHOT1 and PHOT2) (2-4) and has been well characterized (1, 5-7). LOV domaincontaining proteins are distributed not only in plants but also in many other organisms. For example, WC-1 and VVD are found in the ascomycete fungus Neurospora crassa (8), aureochrome in the alga Vaucheria frigida (9), and bacterial histidine kinases in the animal pathogenic bacterium Brucella abortus (10) and in the stalked bacterium Caulobacter crescentus (11). These proteins have all been shown to function as photoreceptors.Six genes encoding LOV domain-containing proteins are found in the genome of Arabidopsis thaliana. Among them, two encode phototropins, and three encode the ADO/FKF/ LKP/ZTL family proteins, proteins involved in circadian clock and photoperiod-dependent flowering functions (12-15). All of the LOV domains of phototropins and ADO/FKF/LKP/ZTL family proteins are shown to bind FMN (2,4,15,16). In addition to those above, there exists a unique LOV domain-containing protein, referred to as PAS/LOV protein (PLP), 2 which contains a PAS domain followed by a LOV domain (1, 17).Ogura et al. (18) screened for proteins interacting with A. thaliana PLP using the yeast two-hybrid system. They isolated VTC2 (vitamin C defective 2), VTC2L (VTC2-like), and BLH10A and BLH10B (BEL1-like homeodomain 10 A and B proteins) as interacting proteins in yeast cells. The molecular interactions between PLP and VTC2L, BLH10A, or BLH10B in yeast were dependent on blue light irradiation, suggesting that PLP may function as a photoreceptor (18). Blue light treatment diminished the interaction. However, the flavin binding and photochemical properties of the A. thaliana PLP are unclear because the recombinant protein expressed in Escherichia coli did not show absorption spectra of flavin-binding proteins (18).In this study, we isolate cDNAs coding for PLP homologs from tomato (Solanum lycopersicum) and the moss Physcomitrella patens, and phylogenetic analysis shows that the PAS domains of the homologs, even the PAS domain of A. thaliana PLP, are closely related to the LOV domain. Thus, we propose that the isolated LOV domain-containing protein should be referred to as LOV/LOV protein (LLP), instead of PLP. Here, we analyze chromophore binding and photochemical properties of LLP from S. lycopersicum using wild-type and mutant proteins expressed in E. coli. We also examine both LOV domains of both PpLLPs for FMN binding and photochemical properties.

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Section: Discussionmentioning

confidence: 99%

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains, LOV/LOV Proteins

Kasahara

Torii

Fujita

et al. 2010

Journal of Biological Chemistry

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“…Dimerization of Phot1-The present SAXS evidence as well as the quaternary structures of LOV1 (15,16) and LOV2-J␣ (17) implies that full-length At phot1 probably exists as a dimer. However, the arrangement of the LOV1 dimer relative to the LOV2 dimer is still unknown.…”

Section: Arrangement Of the Functional Domains In P1l2lk-mentioning

confidence: 91%

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1

Oide

Okajima

Kashojiya

et al. 2016

Journal of Biological Chemistry

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Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygenvoltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2. Phototropin (phot)2 is one of the blue light (BL) receptor proteins in plants (1, 2). It plays several important roles to maximize the efficiency of photosynthesis: namely phototropism (3), chloroplast movement (4, 5), stomata opening (6), and leaf expansion (7). Most plants possess two isoforms of phot, designated as phot1 and phot2 (1). Both isoforms redundantly regulate stomata opening (6) and chloroplast accumulation depending on fluence rate of BL (3). However, only phot2 is responsible for the photoavoidance response in chloroplast relocation (4). Regarding the phototropic response, phot2 works under high irradiance condition, whereas phot1 predominantly initiates the response under low irradiance condition (3).The structure of plant phot, which consists of ϳ950 -1000 amino acid residues and two flavin mononucleotide (FMN) molecules (1), folds into three functional domains (Fig. 1A). The N-terminal half contains two light-oxygen-voltage-sensing domains (LOVs) (designated LOV1 and LOV2) (8, 9), which belong to the PAS (Per-Arnt-Sim) superfamily found in heterodimeric interaction in cellular signaling. Each LOV domain receives BL via the FMN molecule bound to the pocket formed by the characteristic ␣-helix/␤-strand-mixed structure known as the ␣/␤-scaffold (10, 11). The C-terminal half of phot is a serine-threonine kinase (STK) domain classified into group VIII of the AGC family (12). The linker region, which has J␣-heli...

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“…In vitro transphosphorylation between glutathione S-transferase (GST)-tagged and non-GST-tagged versions of phot1 was also observed in insect cells using native ( Figure 10A) or constitutively active (see Supplemental Figure 8 online) forms of the receptor, further demonstrating that phot1 is capable of intermolecular phosphorylation. Isolated LOV1 domains of Arabidopsis phot1 form dimers and are proposed to function as sites of receptor dimerization (Nakasako et al, 2004(Nakasako et al, , 2008Katsura et al, 2009). However, we found that the LOV2K region of phot1 retains light-induced kinase activity when expressed in insect cells and is able to transphosphorylate phot1 D806N ( Figure 10B).…”

Section: Phot1 Internalization Is Promoted By Intermolecular Phosphormentioning

confidence: 99%

“…While LOV1 domains of phot1 form homodimers in vitro (Nakasako et al, 2004(Nakasako et al, , 2008Katsura et al, 2009), the ability of phot1 LOV2K to mediate intermolecular phosphorylation in vitro and in vivo indicates that this mechanism can occur in the absence of LOV1 (Figure 10). The LOV2 domain of phot1 is known to dimerize in solution depending on its concentration (Nakasako et al, 2004;Katsura et al, 2009). Studies are now aimed at defining the site(s) of receptor dimerization and to assess whether intermolecular phosphorylation is essential for phototropin signaling.…”

Section: Intermolecular Autophosphorylation Of Phot1 Is Not Dependentmentioning

confidence: 99%

Domain Swapping to Assess the Mechanistic Basis ofArabidopsisPhototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

et al. 2009

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Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

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Oligomeric structure of LOV domains in Arabidopsis phototropin

Cited by 35 publications

References 23 publications

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains, LOV/LOV Proteins

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains, LOV/LOV Proteins

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1

Domain Swapping to Assess the Mechanistic Basis ofArabidopsisPhototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

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