Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2016; Elsevier BV; Volume: 291; Issue: 38 Linguagem: Inglês

10.1074/jbc.m116.735787

ISSN

1083-351X

Autores

Mao Oide, Koji Okajima, Sachiko Kashojiya, Yuki Takayama, Tomotaka Oroguchi, Takaaki Hikima, Masaki Yamamoto, Masayoshi Nakasako,

Tópico(s)

Photoreceptor and optogenetics research

Resumo

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-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. Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-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) 2The abbreviations used are: phot, phototropin; At, A. thaliana; Cr, C. reinhardtii; BL, blue light; BL1, state under blue light irradiation in small-angle x-ray scattering; D450, dark state; DK1, dark state 1 in small-angle x-ray scattering experiment; DK2, dark state 2 in small-angle x-ray scattering experiment; LOV, light-oxygen-voltage; P1L2LK, phot1 L2LK; phot1, At phototropin1; phot2, At phototropin2; S390, adduct state; SAXS, small-angle x-ray scattering; STK, serine-threonine kinase; L2LK, LOV2-linker-STK fragment; SEC, size exclusion chromatography; PCA, principal component analysis. is one of the blue light (BL) receptor proteins in plants (1Christie J.M. Phototropin blue-light receptors.Annu. Rev. Plant Biol. 2007; 58: 21-45Crossref PubMed Scopus (675) Google Scholar, 2Christie J.M. Reymond P. Powell G.K. Bernasconi P. Raibekas A.A. Liscum E. Briggs W.R. Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.Science. 1998; 282: 1698-1701Crossref PubMed Scopus (523) Google Scholar). It plays several important roles to maximize the efficiency of photosynthesis: namely phototropism (3Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6969-6974Crossref PubMed Scopus (595) Google Scholar), chloroplast movement (4Kagawa T. Sakai T. Suetsugu N. Oikawa K. Ishiguro S. Kato T. Tabata S. Okada K. Wada M. Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.Science. 2001; 291: 2138-2141Crossref PubMed Scopus (550) Google Scholar, 5Jarillo J.A. Gabrys H. Capel J. Alonso J.M. Ecker J.R. Cashmore A.R. Phototropin-related NPL1 controls chloroplast relocation induced by blue light.Nature. 2001; 410: 952-954Crossref PubMed Scopus (383) Google Scholar), stomata opening (6Kinoshita T. Doi M. Suetsugu N. Kagawa T. Wada M. Shimazaki K. Phot1 and phot2 mediate blue light regulation of stomatal opening.Nature. 2001; 414: 656-660Crossref PubMed Scopus (722) Google Scholar), and leaf expansion (7de Carbonnel M. Davis P. Roelfsema M.R. Inoue S. Schepens I. Lariguet P. Geisler M. Shimazaki K. Hangarter R. Fankhauser C. The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning.Plant Physiol. 2010; 152: 1391-1405Crossref PubMed Scopus (134) Google Scholar). Most plants possess two isoforms of phot, designated as phot1 and phot2 (1Christie J.M. Phototropin blue-light receptors.Annu. Rev. Plant Biol. 2007; 58: 21-45Crossref PubMed Scopus (675) Google Scholar). Both isoforms redundantly regulate stomata opening (6Kinoshita T. Doi M. Suetsugu N. Kagawa T. Wada M. Shimazaki K. Phot1 and phot2 mediate blue light regulation of stomatal opening.Nature. 2001; 414: 656-660Crossref PubMed Scopus (722) Google Scholar) and chloroplast accumulation depending on fluence rate of BL (3Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6969-6974Crossref PubMed Scopus (595) Google Scholar). However, only phot2 is responsible for the photoavoidance response in chloroplast relocation (4Kagawa T. Sakai T. Suetsugu N. Oikawa K. Ishiguro S. Kato T. Tabata S. Okada K. Wada M. Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.Science. 2001; 291: 2138-2141Crossref PubMed Scopus (550) Google Scholar). Regarding the phototropic response, phot2 works under high irradiance condition, whereas phot1 predominantly initiates the response under low irradiance condition (3Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6969-6974Crossref PubMed Scopus (595) Google Scholar). The structure of plant phot, which consists of ∼950–1000 amino acid residues and two flavin mononucleotide (FMN) molecules (1Christie J.M. Phototropin blue-light receptors.Annu. Rev. Plant Biol. 2007; 58: 21-45Crossref PubMed Scopus (675) Google Scholar), folds into three functional domains (Fig. 1A). The N-terminal half contains two light-oxygen-voltage-sensing domains (LOVs) (designated LOV1 and LOV2) (8Möglich A. Ayers R.A. Moffat K. Structure and signaling mechanism of Per-ARNT-Sim domains.Structure. 2009; 17: 1282-1294Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 9Möglich A. Yang X. Ayers R.A. Moffat K. Structure and function of plant photoreceptors.Annu. Rev. Plant Biol. 2010; 61: 21-47Crossref PubMed Scopus (402) Google Scholar), 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 α-helix/β-strand-mixed structure known as the α/β-scaffold (10Crosson S. Moffat K. Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2995-3000Crossref PubMed Scopus (426) Google Scholar, 11Crosson S. Moffat K. Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch.Plant Cell. 2002; 14: 1067-1075Crossref PubMed Scopus (324) Google Scholar). The C-terminal half of phot is a serine-threonine kinase (STK) domain classified into group VIII of the AGC family (12Bögre L. Okrész L. Henriques R. Anthony R.G. Growth signalling pathways in Arabidopsis and the AGC protein kinases.Trends Plant Sci. 2003; 8: 424-431Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The linker region, which has Jα-helix lying on the α/β-scaffold in the N-terminal quarter, connects LOV2 and STK (13Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (640) Google Scholar). The LOV1 domain acts as a site for dimerization of Arabidopsis thaliana (At) phot1 and phot2 (14Nakasako M. Iwata T. Matsuoka D. Tokutomi S. Light-induced structural changes of LOV domain-containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle x-ray scattering.Biochemistry. 2004; 43: 14881-14890Crossref PubMed Scopus (87) Google Scholar15Nakasako M. Zikihara K. Matsuoka D. Katsura H. Tokutomi S. Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2.J. Mol. Biol. 2008; 381: 718-733Crossref PubMed Scopus (100) Google Scholar, 16Katsura H. Zikihara K. Okajima K. Yoshihara S. Tokutomi S. Oligomeric structure of LOV domains in Arabidopsis phototropin.FEBS Lett. 2009; 583: 526-530Crossref PubMed Scopus (31) Google Scholar17Halavaty A.S. Moffat K. Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2013; 69: 1316-1321Crossref PubMed Scopus (30) Google Scholar), whereas BL-activated LOV2 is primarily responsible for inducing autophosphorylation of phot (18Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (248) Google Scholar) and phosphorylation of substrate proteins such as ABCB19 (19Christie J.M. Yang H. Richter G.L. Sullivan S. Thomson C.E. Lin J. Titapiwatanakun B. Ennis M. Kaiserli E. Lee O.R. Adamec J. Peer W.A. Murphy A.S. phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism.PLoS Biol. 2011; 9: e1001076Crossref PubMed Scopus (190) Google Scholar), PKS4 (20Demarsy E. Schepens I. Okajima K. Hersch M. Bergmann S. Christie J. Shimazaki K. Tokutomi S. Fankhauser C. Phytochrome kinase substrate 4 is phosphorylated by the phototropin 1 photoreceptor.EMBO J. 2012; 31: 3457-3467Crossref PubMed Scopus (79) Google Scholar), and BLUS1 (21Takemiya A. Sugiyama N. Fujimoto H. Tsutsumi T. Yamauchi S. Hiyama A. Tada Y. Christie J.M. Shimazaki K. Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening.Nat. Commun. 2013; 4: 2094Crossref PubMed Scopus (134) Google Scholar) in the downstream signaling pathways. Structural and spectroscopic studies on phot and its functional domains have focused on the molecular mechanism for how BL-excited phot acquires the kinase activity. Upon BL irradiation, LOV undergoes a characteristic photoreaction cycle (22Salomon M. Christie J.M. Knieb E. Lempert U. Briggs W.R. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.Biochemistry. 2000; 39: 9401-9410Crossref PubMed Scopus (518) Google Scholar, 23Swartz T.E. Corchnoy S.B. Christie J.M. Lewis J.W. Szundi I. Briggs W.R. Bogomolni R.A. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.J. Biol. Chem. 2001; 276: 36493-36500Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar24Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Nagatani A. Briggs W.R. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (256) Google Scholar). A transient adduct is formed between FMN and a conserved Cys residue (S390 intermediate state with an absorption maximum at 390 nm) (22Salomon M. Christie J.M. Knieb E. Lempert U. Briggs W.R. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.Biochemistry. 2000; 39: 9401-9410Crossref PubMed Scopus (518) Google Scholar, 23Swartz T.E. Corchnoy S.B. Christie J.M. Lewis J.W. Szundi I. Briggs W.R. Bogomolni R.A. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.J. Biol. Chem. 2001; 276: 36493-36500Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), which then is thermally broken to the dark state (D450 with an absorption maximum at 450 nm). In the S390 of LOV2, a couple of small structural changes in the side chains of the amino acid residues near FMN are thought to be the primary step necessary for the activation of STK (18Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (248) Google Scholar). Under BL irradiation, the Jα-helix unfolds, and then its interaction with the α/β-scaffold would change (13Harper S.M. Neil L.C. Gardner K.H. Structural basis of a phototropin light switch.Science. 2003; 301: 1541-1544Crossref PubMed Scopus (640) Google Scholar, 25Iwata T. Nozaki D. Tokutomi S. Kagawa T. Wada M. Kandori H. Light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 studied by low-temperature FTIR and UV-visible spectroscopy.Biochemistry. 2003; 42: 8183-8191Crossref PubMed Scopus (97) Google Scholar, 26Harper S.M. Christie J.M. Gardner K.H. Disruption of the LOV-Jα helix interaction activates phototropin kinase activity.Biochemistry. 2004; 43: 16184-16192Crossref PubMed Scopus (252) Google Scholar27Nakasone Y. Eitoku T. Matsuoka D. Tokutomi S. Terazima M. Dynamics of conformational changes of Arabidopsis phototropin 1 LOV2 with the linker domain.J. Mol. Biol. 2007; 367: 432-442Crossref PubMed Scopus (74) Google Scholar). To date, we have been investigating the minimum functional unit of phot, LOV2-linker-STK fragment (L2LK) (28Okajima K. Matsuoka D. Tokutomi S. LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1.FEBS Lett. 2011; 585: 3391-3395Crossref PubMed Scopus (25) Google Scholar, 29Okajima K. Kashojiya S. Tokutomi S. 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.J. Biol. Chem. 2012; 287: 40972-40981Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), to understand how the conformational changes in LOV2 propagate to STK. For example, the BL-induced rearrangement of LOV2 and STK was found in the L2LKs of At phot2 (30Takayama Y. Nakasako M. Okajima K. Iwata A. Kashojiya S. Matsui Y. Tokutomi S. Light-induced movement of the LOV2 domain in an Asp720Asn mutant LOV2-kinase fragment of Arabidopsis phototropin 2.Biochemistry. 2011; 50: 1174-1183Crossref PubMed Scopus (19) Google Scholar) and Chlamydomonas reinhardtii (Cr) phot (31Okajima K. Aihara Y. Takayama Y. Nakajima M. Kashojiya S. Hikima T. Oroguchi T. Kobayashi A. Sekiguchi Y. Yamamoto M. Suzuki T. Nagatani A. Nakasako M. Tokutomi S. 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.J. Biol. Chem. 2014; 289: 413-422Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 32Okajima K. Molecular mechanism of phototropin light signaling.J. Plant Res. 2016; 129: 149-157Crossref PubMed Scopus (23) Google Scholar) through structural studies by using small-angle x-ray scattering (SAXS). Mutation studies demonstrated that amino acid residues critical to the phosphorylation activity of STK are localized around the N-terminal regions of LOV2 (33Aihara Y. Yamamoto T. Okajima K. Yamamoto K. Suzuki T. Tokutomi S. Tanaka K. Nagatani A. Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.J. Biol. Chem. 2012; 287: 9901-9909Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 34Kashojiya S. Okajima K. Shimada T. Tokutomi S. Essential role of the A′α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.PLoS One. 2015; 10: e0124284Crossref PubMed Scopus (13) Google Scholar). From these studies, we envision a scenario whereby the linker and its adjoining region mediate the propagation of small conformational changes in BL-excited LOV2 to STK (Fig. 1B). In the case of At phot1, mutagenesis studies of phot1 L2LK (P1L2LK) (residues 449–996; Fig. 1A) are in progress to identify the residues necessary for the BL-dependent kinase activity (34Kashojiya S. Okajima K. Shimada T. Tokutomi S. Essential role of the A′α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.PLoS One. 2015; 10: e0124284Crossref PubMed Scopus (13) Google Scholar, 35Kashojiya S. Yoshihara S. Okajima K. Tokutomi S. The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor.FEBS Lett. 2016; 590: 139-147Crossref PubMed Scopus (7) Google Scholar). Cys-512 residue in LOV2 is critical for the adduct formation with FMN in the photoreaction cycle. Asp-806 residue in STK is essential for the kinase activity (18Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function.Plant J. 2002; 32: 205-219Crossref PubMed Scopus (248) Google Scholar). Lys-475 residue in the A′α/Aβ gap of the N terminus of LOV2 and Lys-636 residue in the linker region would have important roles in the BL-dependent kinase activity. Although the arrangement of LOV2 and STK in phot1 remains unknown, the dimer structures of LOV1 and LOV2 are now known (Fig. 1C) (14Nakasako M. Iwata T. Matsuoka D. Tokutomi S. Light-induced structural changes of LOV domain-containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle x-ray scattering.Biochemistry. 2004; 43: 14881-14890Crossref PubMed Scopus (87) Google Scholar15Nakasako M. Zikihara K. Matsuoka D. Katsura H. Tokutomi S. Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2.J. Mol. Biol. 2008; 381: 718-733Crossref PubMed Scopus (100) Google Scholar, 16Katsura H. Zikihara K. Okajima K. Yoshihara S. Tokutomi S. Oligomeric structure of LOV domains in Arabidopsis phototropin.FEBS Lett. 2009; 583: 526-530Crossref PubMed Scopus (31) Google Scholar17Halavaty A.S. Moffat K. Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2013; 69: 1316-1321Crossref PubMed Scopus (30) Google Scholar). Therefore, the structural roles of the lysine residues are difficult to ascertain. Here, we study the molecular structures of wild-type and mutated P1L2LK by SAXS to better understand the molecular mechanism responsible for BL-induced kinase activity of phot1. The purity of the wild-type P1L2LK was greater than 98% as indicated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. Size exclusion chromatography (SEC) indicated that P1L2LK probably forms a dimer in solution in the concentration range used in the spectroscopic and SAXS measurements (0.5–2.3 mg ml−1) (Fig. 2A). The absorption spectra of D450 and S390 states were almost identical to those of the recombinant phot1 LOV2 (24Kasahara M. Swartz T.E. Olney M.A. Onodera A. Mochizuki N. Fukuzawa H. Asamizu E. Tabata S. Kanegae H. Takano M. Christie J.M. Nagatani A. Briggs W.R. Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii.Plant Physiol. 2002; 129: 762-773Crossref PubMed Scopus (256) Google Scholar). Under successive BL irradiation, the amount of accumulated S390 depended on the fluence of BL because of the dark (thermal) reversion from S390 to D450. For SAXS measurements, almost all of the P1L2LK in solution sample must be converted to the S390 state under BL irradiation. To estimate the fluence rate necessary to accumulate almost completely S390, we measured the amount of S390 under BL irradiation by using our custom-made cell holder mounted on the UV-visible spectrometer. This device is limited to measuring the UV-visible spectra under a fluence rate of less than 200 μmol m−2 s−1 because of the stray light under high fluence rate. Therefore, we measured the amount of S390 under a fluence rate of less than 200 μmol m−2 s−1. Then the fluence rate necessary for the almost complete accumulation of S390 was estimated to be 400 μmol m−2 s−1 by an extrapolation (see Fig. 2B, inset). Within 120 s after starting BL irradiation of 200 μmol m−2 s−1, the amount of L2LK in S390 reached saturation. The thermal dark reversion took more than 400 s after turning off BL at 293 K (Fig. 2C). After BL irradiation of 200–400 μmol m−2 s−1, P1L2LK undergoes photoreaction. This result indicated little molecular damage in P1L2LK under the BL irradiation of the fluence rate of 400 μmol m−2 s−1. Little x-ray radiation damage to the samples was found as verified by very small differences between the absorption spectra and SDS-PAGE patterns of specimens before and after the exposure to x-rays within one round of DK1-BL1-DK2 exposures. To avoid x-ray radiation damage during several rounds of x-ray exposure, SAXS profiles of the three states were recorded through only one round of exposure as done in our previous SAXS studies on the LOV2-linker-kinase fragment of At phot2 (30Takayama Y. Nakasako M. Okajima K. Iwata A. Kashojiya S. Matsui Y. Tokutomi S. Light-induced movement of the LOV2 domain in an Asp720Asn mutant LOV2-kinase fragment of Arabidopsis phototropin 2.Biochemistry. 2011; 50: 1174-1183Crossref PubMed Scopus (19) Google Scholar) and full-length phot of Chlamydomonas (31Okajima K. Aihara Y. Takayama Y. Nakajima M. Kashojiya S. Hikima T. Oroguchi T. Kobayashi A. Sekiguchi Y. Yamamoto M. Suzuki T. Nagatani A. Nakasako M. Tokutomi S. 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.J. Biol. Chem. 2014; 289: 413-422Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For each P1L2LK solution, we sequentially recorded the SAXS profiles of P1L2LK in the order of DK1, BL1, and DK2 states (Fig. 3A). The scattering intensities of BL1 increased in S < 0.003 Å−1 but decreased in 0.004 < S < 0.010 Å−1 for the DK1 state (Fig. 3A). The SAXS profiles of the DK2 state resembled those of the DK1 state rather than the BL1 state. The profile difference, calculated by subtracting the profile of the BL1 state from that of the DK1 state, is negative in S < 0.003 Å−1 with a maximum at approximately S = 0.005 Å−1. The profile difference obtained by subtracting the profile of the BL1 state from that of the DK2 state is positive in S < 0.003 Å−1 with a minimum at approximately S = 0.005 Å−1. Because these two cases are almost opposite in sign, the SAXS changes reflecting BL-induced conformational alternation of P1L2LK were probably photoreversible. When taking into consideration the reciprocity between the length of the scattering vector and the distance between any pairs of large electron density clusters, such as functional domains, the changes in 0.004 < S < 0.010 Å−1 suggest rearrangement of domains separated by ∼150 Å. In this regard, differences in the P(r) functions between the DK1 and BL1 states were prominent at r > 100 Å (Fig. 3C). The Guinier plots for SAXS profiles of P1L2LK in the DK1, BL1, and DK2 states could be approximated by straight lines in S2 < 40 × 10−6 Å−2 (Fig. 3D). The calculated C/I(S = 0, C) and Rg2(C) values displayed almost linear concentration dependences (Fig. 3E), indicating their monodispersive properties. The apparent molecular weight estimated from I(S = 0, C = 0) indicated that P1L2LK forms a dimer both in the dark and under BL. This result is consistent with the crystal structure of LOV2-Jα (17Halavaty A.S. Moffat K. Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2013; 69: 1316-1321Crossref PubMed Scopus (30) Google Scholar) and the SEC prior to the SAXS measurements. Because the I(S = 0, C = 0) value of the DK1 state is almost the same as that of the BL1 state, the dimer structure is kept under BL irradiation. The P1L2LK in the BL1 state had an Rg(C = 0) of 48.8 ± 0.5 Å that was significantly larger than that of either the DK1 date (42.9 ± 0.5 Å) or the DK2 state (43.5 ± 0.5 Å). This increase in Rg(C = 0) suggests an expansion and/or hydration changes in P1L2LK under BL. The slightly greater Rg(C = 0) of the DK2 state than that of the DK1 state indicates that the thermal reversion of the overall structure proceeded more slowly than did the structural relaxation around the FMN monitored in the absorption spectra (Fig. 2B). Each molecular shape, restored by applying the GASBOR program to the observed SAXS profile, gave a theoretical scattering curve that closely matched those observed with χ2 values of 2% (Fig. 4A). Following the principal component analysis (PCA) and K-means clustering analysis, the molecular shapes of DK1, BL1, and DK2 were calculated from 472, 380, and 307 restored models, respectively. The averaged molecular shape of P1L2LK in the DK1 state appeared as two bean-shaped lobes in a twisted arrangement along the long molecular axes (Fig. 4B). Its dimensions are 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape of the BL1 state was slightly different from that of the DK1 state. The two lobes were elongated and simultaneously tilted by ∼30° against the 2-fold symmetry axis of the dimer. Then the dimension along the molecular axis was enlarged by ∼20 Å from that of the DK1 state. The molecular shape and dimensions of P1L2LK in the DK2 state are more similar to those of the DK1 state than of the BL1 state. Structural roles of Lys-475 and Lys-636 residues located at the interface between LOV2 and STK were studied through SAXS for K475A/D806N and K636A/D806N double mutants. K475A and K636A mutations cause reduction of the kinase activity of STK (34Kashojiya S. Okajima K. Shimada T. Tokutomi S. Essential role of the A′α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.PLoS One. 2015; 10: e0124284Crossref PubMed Scopus (13) Google Scholar, 35Kashojiya S. Yoshihara S. Okajima K. Tokutomi S. The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor.FEBS Lett. 2016; 590: 139-147Crossref PubMed Scopus (7) Google Scholar). D806N mutation results in the loss of the phosphorylation activity of STK because the side chain of the Asp-806 residue is expected to stabilize the position of a magnesium ion necessary for the ATPase activity as well as various kinases (36Hanks S.K. Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification.FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2319) Google Scholar). Therefore, the D806N mutation was introduced to avoid heterogeneity of P1L2LK in regard to phosphorylation in the purification (29Okajima K. Kashojiya S. Tokutomi S. 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.J. Biol. Chem. 2012; 287: 40972-40981Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). As a reference, the C512A/D806N mutant was used to examine whether BL irradiation is essential for structural changes because C512A mutation results in the loss of adduct formation capability under BL irradiation. Prior to SAXS measurements, the dark reversion kinetics of D806N, K475A/D806N, and K636A/D806N mutants were determined through UV-visible absorption measurements. Prior to the x-ray measurements, the relaxation times of the S390 states of D806N, K475A/D806N, and K636A/D806N were determined by measuring their UV-visible absorption. The re

Referência(s)