Structural and Biochemical Characterization of a Cyanobacterium Circadian Clock-modifier Protein
2006; Elsevier BV; Volume: 282; Issue: 2 Linguagem: Inglês
10.1074/jbc.m608148200
ISSN1083-351X
AutoresKyohei Arita, Hiroshi Hashimoto, Kumiko Igari, Mayuko Akaboshi, Shinsuke Kutsuna, Mamoru Sato, Toshiyuki Shimizu,
Tópico(s)Circadian rhythm and melatonin
ResumoCircadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an (α + β) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor. Circadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an (α + β) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor. Organisms from cyanobacteria to mammals have a circadian rhythm, an adaptation to diurnal environmental changes such as light and temperature. The timing machinery of the rhythm is known as the “circadian clock” (1Bünning E. The Physiological Clock. Springer-Verlag, Berlin1973Google Scholar, 2Dunlap J.C. Loros J.J. DeCoursey P.J. Chronobiology: Biological Timekeeping. Sinauer Associates Inc.,, Sunderland, MA2004Google Scholar). The clock has three representative properties (free running, resetting, and temperature compensation), which are essential to a system of time measurement. In cyanobacteria, circadian rhythms have been reported in amino acid uptake (3Chen T. Chen T.L. Hung L.M. Hung T.C. Plant Physiol. 1991; 97: 55-59Crossref PubMed Scopus (81) Google Scholar), cell division (4Mori T. Binder B. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10183-10188Crossref PubMed Scopus (260) Google Scholar, 5Kondo T. Mori T. Lebedeva N.V. Aoki S. Ishiura M. Golden S.S. Science. 1997; 275: 224-227Crossref PubMed Scopus (135) Google Scholar), and various gene expressions (6Liu Y. Tsinoremas N.F. Johnson C.H. Lebedeva N.V. Golden S.S. Ishiura M. Kondo T. Genes Dev. 1995; 9: 1469-1478Crossref PubMed Scopus (307) Google Scholar, 7Nakahira Y. Katayama M. Miyashita H. Kutsuna S. Iwasaki H. Oyama T. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 881-884Crossref PubMed Scopus (109) Google Scholar, 8Onai K. Morishita M. Itoh S. Okamoto K. Ishiura M. J. Bacteriol. 2004; 186: 4972-4977Crossref PubMed Scopus (45) Google Scholar) at each particular time. After a photosynthesis gene (psbAI) in a unicellular strain, Synechococcus elongatus PCC 7942, was shown to be transcribed rhythmically with a circadian period under constant conditions (9Kondo T. Strayer C.A. Kulkarni R.D. Taylor W. Ishiura M. Golden S.S. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5672-5676Crossref PubMed Scopus (387) Google Scholar), more than 100 mutants with defects, including arrhythmia and rhythms with a long or short period, were isolated (10Kondo T. Tsinoremas N.F. Golden S.S. Johnson C.H. Kutsuna S. Ishiura M. Science. 1994; 266: 1233-1236Crossref PubMed Scopus (255) Google Scholar). The mutations were mapped to a gene cluster named kaiABC (11Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar), which is transcribed as kaiA and kaiBC mRNAs by the respective kaiA and kaiBC promoters. Transcription of either the kaiA or kaiBC operon is under circadian feedback regulation, whereby the kaiA and kaiC proteins (KaiA and KaiC) activate and repress the promoter of kaiBC (11Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar), and this feedback functions to maintain normal circadian sustainability, but not the period (12Kutsuna S. Nakahira Y. Katayama M. Ishiura M. Kondo T. Mol. Microbiol. 2005; 57: 1478-1484Crossref Scopus (22) Google Scholar). KaiC has autokinase activity essential to the rhythm, where the kinase reaction is accelerated and decelerated by KaiA and kaiB protein (KaiB), respectively (13Nishiwaki T. Satomi Y. Nakajima M. Lee C. Kiyohara R. Kageyama H. Kitayama Y. Temamoto M. Yamaguchi A. Hijikata A. Go M. Iwasaki H. Takao T. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13927-13932Crossref PubMed Scopus (172) Google Scholar, 14Xu Y. Mori T. Pattanayek R. Pattanayek S. Egli M. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13933-13938Crossref PubMed Scopus (122) Google Scholar, 15Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (239) Google Scholar). The phosphorylation of KaiC recurs with a circadian period in vivo (16Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15788-15793Crossref PubMed Scopus (245) Google Scholar, 17Tomita J. Nakajima M. Kondo T. Iwasaki H. Science. 2005; 307: 251-254Crossref PubMed Scopus (392) Google Scholar), which can be reconstituted in vitro (18Nakajima M. Imai K. Ito H. Nishiwaki T. Murayama Y. Iwasaki H. Oyama T. Kondo T. Science. 2005; 308: 414-415Crossref PubMed Scopus (814) Google Scholar). This observation indicates that various heteromultimeric complexes of KaiA, KaiB, and KaiC are formed in the cell (15Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (239) Google Scholar, 19Kageyama H. Kondo T. Iwasaki H. J. Biol. Chem. 2003; 278: 2388-2395Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In cyanobacteria, the clock is reset by a light or dark pulse (20Aoki S. Kondo T. Wada H. Ishiura M. J. Bacteriol. 1997; 179: 5751-5755Crossref PubMed Google Scholar, 21Schmitz O. Katayama M. Williams S.B. Kondo T. Golden S.S. Science. 2000; 289: 765-768Crossref PubMed Scopus (226) Google Scholar). Resetting-related mutants have been isolated and found to lack normal circadian resetting. Period extender (pex), mutants of which show a short-period phenotype (22Kutsuna S. Kondo T. Aoki S. Ishiura M. J. Bacteriol. 1998; 180: 2167-2174Crossref PubMed Google Scholar), was recently classified as a resetting-related gene (23Takai N. Ikeuchi S. Manabe K. Kutsuna S. J. Biol. Rhythms. 2006; 21: 235-244Crossref PubMed Scopus (23) Google Scholar). Insertion of pex into the genome of the clock mutant C22a (which has a kaiA1 mutation) causes extension of the 22-h period phenotype of C22a to 24 h, similar to that of the wild type (10Kondo T. Tsinoremas N.F. Golden S.S. Johnson C.H. Kutsuna S. Ishiura M. Science. 1994; 266: 1233-1236Crossref PubMed Scopus (255) Google Scholar, 11Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). Levels of pex mRNA and pex protein (Pex) increase in the dark period, and a pex mutant subjected to diurnal light-dark cycles shows an advance in the phase of the rhythm by 3 h, suggesting that Pex has a resetting function (23Takai N. Ikeuchi S. Manabe K. Kutsuna S. J. Biol. Rhythms. 2006; 21: 235-244Crossref PubMed Scopus (23) Google Scholar). Sequence analysis demonstrates that Pex has a PadR domain, which is conserved among PadR proteins in lactobacilli Pediococcus pentosaceus and Pediococcus plantarum (24Barthelmebs L. Lecomte B. Divies C. Cavin J.F. J. Bacteriol. 2000; 182: 6724-6731Crossref PubMed Scopus (88) Google Scholar). PadR is a transcriptional regulator related to multiple antibiotic resistance repressor (MarR) 3The abbreviations used are: MarR, multiple antibiotic resistance repressor; EMSA, electrophoretic mobility shift assay; dsDNA, double-stranded DNA; SeMet, selenomethionine; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RTP, replication terminator protein. family proteins with DNA-binding activity (25Nikaido H. Curr. Opin. Microbiol. 1998; 5: 516-523Crossref Scopus (252) Google Scholar), and binds to the promoter of the padA gene, which is essential for metabolizing environmental toxins such as p-coumaric acid. Despite these structural indications, however, no experimental evidence has been obtained regarding the function of Pex. To understand the molecular mechanism involved in regulating the circadian clock oscillation in cyanobacteria, we have determined the crystal structure of a N-terminal deletion mutant of Pex, Pex-(15–148), from S. elongatus PCC 7942 at 1.8-Å resolution by x-ray diffraction. To our knowledge, this is the first structure determination of a protein of the circadian input system in the cyanobacteria circadian clock. Pex-(15–148) has a winged-helix motif and is likely to function as a dimer with a unique subunit arrangement. Electrophoresis mobility shift assay (EMSA) and mutation analysis demonstrate that Pex has specific affinity for double-stranded DNA (dsDNA) containing the kaiA upstream site and that the wing region in the winged-helix motif has a crucial role in interaction with the DNA. Protein Expression and Purification—Genes encoding full-length Pex, Pex-(1–148), and its N-terminal deletion mutants (Pex-(15–148) and selenomethionine (SeMet)-labeled Pex-(15–148)) were subcloned into expression vector pGEX6P-1 (GE Healthcare) at the 5′ BamHI-XhoI site, and expressed as fusion proteins of glutathione S-transferase (GST) in Escherichia coli strain BL21(DE3) for Pex-(1–148) and Pex-(15–148), and in E. coli strain B834(DE3) for SeMet-labeled Pex-(15–148). The cells were grown at 37 °C to a cell density of 0.4–0.6 at 660 nm and incubated for a further 5 h after the addition of 0.1 mm isopropyl β-d-thiogalactopyranoside at 18 °C. Pex-(1–148), Pex-(15–148), and SeMet-labeled Pex-(15–148) were purified under the same experimental conditions. The cells were harvested, resuspended in lysis buffer (20 mm HEPES buffer (pH 7.0) containing 0.1 m NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and 0.5 mm phenylmethylsulfonyl fluoride) and disrupted by sonication at 4 °C. After centrifugation, the supernatant was applied to a GST affinity column of glutathione-Sepharose 4B (GS4B) (GE Healthcare) equilibrated with phenylmethylsulfonyl fluoride-free lysis buffer. The adsorbed fraction was eluted with elution buffer (80 mm Tris-HCl buffer (pH 8.5) containing 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and 30 mm reduced glutathione). The fusion protein was cleaved by 100 units of PreScission Protease (GE Healthcare) for 40 h at 4 °C. The cleaved protein was applied to an affinity HiTrap heparin column (GE Healthcare), equipped with anÁKTA Prime system (GE Healthcare) equilibrated at 4 °C with 20 mm Tris-HCl buffer (pH 8.0) containing 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 10% glycerol. The adsorbed fraction was eluted with a linear gradient from 100 to 500 mm NaCl at 4 °C. Homogeneity of the purified protein was confirmed by SDS-PAGE. Purified protein in 10 mm Tris-HCl buffer (pH 8.0) containing 200 mm NaCl, 1 mm dithiothreitol, and 1 mm EDTA was concentrated at 4 °C to ∼4 mg/ml in a Centricon-10 concentrator (Amicon). The concentration of the purified protein was determined by UV absorption. Crystallization and Data Collection—Pex-(15–148) was successfully crystallized at 20 °C by the hanging drop vapor-diffusion method using 0.5–1.0 m Li2SO4 as a precipitant in 0.1 m sodium acetate buffer (pH 3.8–4.6) containing 50 mm magnesium acetate. Crystals of SeMet-labeled Pex-(15–148) were obtained under the same conditions. All x-ray diffraction data were collected at 100 K on beamline BL-5 at Photon Factory, Tsukuba, Japan, using an ADSC Quantum 315 CCD detector. Before the x-ray experiments, crystals of Pex-(15–148) and SeMet-labeled Pex-(15–148) were each soaked in the crystallization buffer containing 20% ethylene glycol as a cryoprotectant. Diffraction data were processed with HKL2000 (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The crystallographic data and data collection statistics of Pex-(15–148) and SeMet-labeled Pex-(15–148) are given in Table 1.TABLE 1Crystallographic data, data collection, and refinement statisticsSeMet-labeled Pex-(15—148)Pex-(15—148)EdgePeakHigh remoteLow remoteCrystallographic data and data collection statisticsWavelength (Å)0.979200.979310.964080.983191.03973Space groupP212121P212121a (Å)56.757.2b (Å)61.561.6c (Å)75.175.7Resolution (Å)50.0-1.850.0-1.850.0-1.850.0-1.832.2-1.8Total observations152,594153,135153,211153,165178,124Unique reflections24,16524,19324,23724,21224,970R merge (%)aValues in parentheses are for highest resolution shell. The resolution ranges of their outer shells are 1.86-1.80 Å for native data and 1.86-1.80 Å for Se-derivative data,bRmerge = Σ|I — (I)|/ΣI; calculated for all data5.9 (50.4)6.7 (50.9)5.2 (50.8)4.3 (50.1)5.8 (42.4)Completeness (%)aValues in parentheses are for highest resolution shell. The resolution ranges of their outer shells are 1.86-1.80 Å for native data and 1.86-1.80 Å for Se-derivative data97.2 (75.8)97.3 (77.0)97.3 (77.4)97.1 (75.1)98.2 (85.3)Redundancy (%)aValues in parentheses are for highest resolution shell. The resolution ranges of their outer shells are 1.86-1.80 Å for native data and 1.86-1.80 Å for Se-derivative data6.3 (3.1)6.4 (3.2)6.4 (3.2)6.4 (3.2)7.2 (4.1)I/σ (I)12.412.512.412.519.0Phasing poweriso (acen/cen)cPhasing power = root mean square heavy atom structure factor/residual lack of closure1.26/0.841.98/1.251.74/1.11RCullis (ano/iso)dRCullis = Σ||FPH — FP| — | FH (calc) ||/|FPH — FP|0.55/0.750.66/0.600.97/0.64Mean figure of merit0.69Refinement statisticsResolution range (Å)32.2-1.80R (%)eR and Rfree = Σ||Fo| — | Fc||/|Fp|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement19.6Rfree (%)eR and Rfree = Σ||Fo| — | Fc||/|Fp|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement23.4Mean B factor (Å2)33.3Root mean square deviationsBond length (Å)0.015Bond angle (°)1.461No. of atomsProtein1900Water molecules144SO42— ions10 (2 SO42—)a Values in parentheses are for highest resolution shell. The resolution ranges of their outer shells are 1.86-1.80 Å for native data and 1.86-1.80 Å for Se-derivative datab Rmerge = Σ|I — (I)|/ΣI; calculated for all datac Phasing power = root mean square heavy atom structure factor/residual lack of closured RCullis = Σ||FPH — FP| — | FH (calc) ||/|FPH — FP|e R and Rfree = Σ||Fo| — | Fc||/|Fp|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement Open table in a new tab Structure Determination and Refinement—No successful solutions were obtained by the molecular replacement method, in which the structures of MarR family proteins were used as search models by Molrep (27Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) and Phaser (28Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1373-1382Crossref PubMed Scopus (789) Google Scholar). This is attributable to significantly large differences in the ternary structures between Pex-(15–148) and the search models. The structure of Pex-(15–148) was therefore solved by multiwavelength anomalous diffraction using the SeMet-labeled Pex-(15–148) crystal. Experimental phases were calculated up to 2.0-Å resolution with SOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) and improved by solvent-flattening with RESOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). An initial model was build by ARP/WARP (30Lamzin V.S. Perrakis A. Wilson K.S. Int. Tables Crystallogr. 2001; F: 720-722Google Scholar), followed by O (31Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), and refined with CNS (32Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). After several cycles of rebuilding with program O and refinement with CNS and REFMAC5 (33Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar), the model finally converged, resulting in a crystallographic R value of 19.6% and a free R value of 23.4% for all diffraction data up to 1.8-Å resolution. The Ramachandran plot of the final model, containing 222 amino acid residues, 144 water molecules, and 2 SO42- ions, shows that all of the amino acid residues have good stereochemistry, with 95.0% of residues in the most favorable regions and 5.0% in additional allowed regions defined by the program PROCHECK (34Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 282-291Crossref Google Scholar). The final refinement statistics are summarized in Table 1. The figures were generated by PyMol (35DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Coordinates for Pex-(15–148) have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics (Protein Data Bank code 2E1N). Site-directed Mutagenesis and EMSA—Alanine mutants of Pex-(1–148), Y82A, T83A, K86A, D90A, R104A, R106A, and R108A, were prepared using pGEX6P-1 wild-type Pex-(1–148) as a template with a QuikChange site-directed mutagenesis kit (Stratagene), according to the manufacturer's recommendations, and confirmed by DNA sequencing. The mutants were expressed as fusion proteins of GST from a pGEX6P-1 vector in E. coli strain BL21(DE3), and purified by the same protocol used for wild-type Pex-(15–148), except that a HiTrap Q column (GE Healthcare) was used instead of the HiTrap heparin column. The DNA binding capacities of wild-type and mutant Pex were examined by EMSA using a 25-bp complementary oligo-nucleotide containing the kaiA upstream sequence including the -60 to -36 region (kaiA upstream site), 5′-ATTTTTCCTTTGTCCAGAGATTAAT-3′ (as the 1st codon of kaiA is +1), which was purchased from Invitrogen. The dsDNA was prepared by annealing at a 1:1 molar ratio in 0.1 m KCl solution. Each solution (5 μl) containing 5 pmol of DNA oligomer (1 μm final concentration) and a 1.5-fold excess of each of wild-type and mutant Pex was separated by electrophoresis at 4 °C on a native 6% polyacrylamide gel at 100 V for 90 min. The running solution was 40 mm Tris-HCl buffer (pH 7.0) containing 20 mm acetic acid and 1 mm EDTA. The shifted bands stained by ethidium bromide in the gel were detected by an Image Analyzer (TOYOBO FAS-III). We also purchased the 25-bp DNAs of a 5-nucleotide downstream sequence (kaiA-1) and 21-nucleotide upstream sequence (kaiA-2)ofthe kaiA upstream site (5′-TCCTTTGTCCAGAGATTAATCTGTC-3′; kaiA-1,5′-TGCAGTGCTAGGCTAAATTAAATTT-3′; kaiA-2) and CmpR binding site in the psbAII promoter region (psbAII) (40 bp, 5′-AGTCCTTAGTTGAACTATTTACGAGACTTAATAGCCTCGT-3′) (45Takahashi Y. Yamaguchi O. Omata T. Mol. Microbiol. 2004; 52: 837-845Crossref PubMed Scopus (35) Google Scholar). In Vivo Rhythm Assay—The bioluminescence reporter S. elongatus PCC 7942 cell NUC42 was used as wild-type, in which a gene fusion of a kaiBC promoter and a gene set of bacterial luciferase, luxAB, was recombined into a genomic region, neutral site I (12Kutsuna S. Nakahira Y. Katayama M. Ishiura M. Kondo T. Mol. Microbiol. 2005; 57: 1478-1484Crossref Scopus (22) Google Scholar). The pex-deficient mutant cell, YCC19, derived from the kaiBC reporter was used (23Takai N. Ikeuchi S. Manabe K. Kutsuna S. J. Biol. Rhythms. 2006; 21: 235-244Crossref PubMed Scopus (23) Google Scholar). The pex-deficient mutant harboring the pex with an E. coli inducible promoter, Ptrc, in the genomic region neutral site II was used as the constitutive induction cell of pex. Then, we constructed the plasmid by which Pex(R106A) protein was expressed with Ptrc inducible promoter. At first we amplified the DNA fragment of the pex gene with base pair substitutions at the 106th codon for Arg to Ala by PCR with KOD DNA polymerase (TOYOBO). The amplified pex DNA was digested with the BamHI restriction enzyme and inserted into unique BamHI site downstream of the Ptrc promoter in the pTS2KPtrc plasmid. The obtained plasmid was checked by sequencing and then introduced into YCC19. These cyanobacterial cells were cultivated on BG-11 agar medium containing 0.1 mm isopropyl β-d-thiogalactopyranoside under continuous light until the colonies with 0.2 mm in diameter appeared. After resetting of the clock by treatment of 12-h darkness, a lid of a microcentrifuge tube containing 30 μl of vacuum pump oil with 3% bioluminescence substrate (n-decanal; Wako) was placed on the agar medium with the colonies. Then, bioluminescence were monitored under continuous light at 30 °C using an automated system equipped with photon-multiplier tubes, as previously described (11Ishiura M. Kutsuna S. Aoki S. Iwasaki H. Andersson C.R. Tanabe A. Golden S.S. Johnson C.H. Kondo T. Science. 1998; 281: 1519-1523Crossref PubMed Scopus (594) Google Scholar). Western Blotting Analysis—About 5000 of the Synechococcus colonies of each cell were formed on BG-11 agar medium containing 0.1 mm isopropyl β-d-thiogalactopyranoside for 4 days in a continuous light condition. Then those colonies were subjected to 12 h dark. The wild-type and pex-deficient mutant colonies subjected to 6 h dark were collected with a spreader. After the 12-h dark treatment, colonies were illuminated for 6 h, then, collected. The cell pellet was suspended in 200 μlof Tris-buffered saline and sonicated to extract soluble protein as described previously (23Takai N. Ikeuchi S. Manabe K. Kutsuna S. J. Biol. Rhythms. 2006; 21: 235-244Crossref PubMed Scopus (23) Google Scholar). The cell extract containing 18 μgof total protein was electrophoresed through a 20% SDS-acrylamide gel and blotted onto a polyvinylidene difluoride membrane. The membrane was blocked in phosphate-buffered saline-Tween (0.3%; PBS-T) for 1 h. The anti-GST-Pex antiserum was diluted 100-fold in PBS-T and used as primary antibody for 1 h. After washing the membrane in PBS-T, horseradish peroxidase-linked anti-rabbit Ig was diluted in PBS-T (1/1000) and used as the secondary antibody (GE Healthcare). These procedures were performed at room temperature except the blotting at 5 °C. The chemiluminescence substrate was Immun-Star HRP Luminol/Enhancer, used with the Fluor-S MultiImager chemiluminescence imaging system (Bio-Rad). Crystal Structure—The crystal structure of Pex-(15–148) from cyanobacterium S. elongatus PCC 7942 was successfully solved by the multiwavelength anomalous diffraction method and refined at 1.8-Å resolution. The molecule has an (α + β) structure, which forms a winged-helix motif together with two additional α helices in the N-terminal (α0 helix, residues 25–33) and C-terminal (α4 helix, residues 120–131) regions (Fig. 1A). The winged-helix motif consists of helices α1 (residues 42–53), α2 (residues 60–70), and α3 (residues 78–90), strands β1 (residues 94–99) and β2 (residues 108–113), and the wing region that connects strands β1 and β2. The amino acid sequence of the molecule, together with assignments of the secondary structure elements, is shown in Fig. 2.FIGURE 2Sequence alignment of cyanobacterial Pex proteins. Residues conserved among the three proteins are shown on a black background. The secondary structure elements, α-helices (bars) and β-strands (arrows), and disordered regions (broken lines) of Pex (15–148) from S. elongatus (molecule B) are indicated above the alignment and colored as described in the legend to Fig. 1. Asterisks indicate the mutation sites used for EMSA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two molecules (molecules A and B), which are related by a non-crystallographic 2-fold axis, are present in the crystal asymmetric unit (Fig. 3A). They have essentially the same structure with root mean square deviations of 1.1 Å for Cα atoms from residues 23 to 132. Residues 15–22 and 133–148 in molecule A and residues 15–22 and 135–148 in molecule B were disordered. Molecule A is in close contact with molecule B, forming a dimer in the crystal. The contact is hydrophobic in nature, with an interface of 1,150 Å2 between helix α4 (Leu124, Leu127, and Tyr131) in molecule A and helix α0 (Met23, Phe25, Ile28, Tyr29, Phe31, and Phe32) in molecule B (Fig. 3B). In addition to these structural features, the Fo - Fc map showed a higher peak with a contour level of more than 13 σ near the wing region (Fig. 1, A and B). On the basis of the chemical components of the crystallization solution and the fact that the peak was near basic residues such as Arg104, Arg106, and Arg108 in the wing region (Fig. 1B), this peak was assigned to SO42-. Interestingly, these three arginine residues are completely conserved among all cyanobacterial Pex proteins (Fig. 2). Comparison of Pex with Other Winged-helix Proteins—Pfam (36Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) predicts that Pex belongs to the PadR protein family, which is related to the MarR family of proteins. In fact, a search of the Protein Data Bank using the Dali server (37Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) showed that Pex is similar to replication terminator protein (RTP) of Bacillus subtilis (PDB code 1BM9, Z-score 10.0) (38Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (76) Google Scholar), metallothionein repressor (SmtB) of Synechococcus PCC 7942 (PDB code 1SMT, Z-score 9.0) (39Cook W.J. Kar S.R. Taylor K.B. Hall L.M. J. Mol. Biol. 1998; 275: 337-346Crossref PubMed Scopus (125) Google Scholar), methicillin repressor (MecI) of Staphylococcus aureus (PDB code 1OKR, Z-score 8.8) (40Garcia-Castellanos R. Marrero A. Mallorqui-Fernandez G. Potempa J. Coll M. Gomis-Ruth F.X. J. Biol. Chem. 2003; 278: 39897-39905Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and MarR of E. coli (PDB code 1JGS, Z-score 8.3) (41Alekshun M.N. Levy S.B. Mealy T.R. Seaton B.A. Head J.F. Nat. Struct. Biol. 2001; 8: 710-714Crossref PubMed Scopus (314) Google Scholar). Although their amino acid sequences are not homologous to that of Pex (sequence identity 10–19%), these proteins all have winged-helix motifs for DNA binding. The winged-helix motifs of Pex, RTP, SmtB, MecI, and MarR could be superimposed with root mean square deviations ranging from 2.4 to 4.0 Å. There is a large difference in a linker region between the α2 and α3 helices. The linker of Pex is significantly longer than those of the other four proteins and thus Asn73–Arg75 in this long linker of Pex can interact with His38–Leu40 in the N-terminal portion that precedes the winged-helix motif (Fig. 1, A and C). This interaction is not observed in the other four proteins due to conformational differences in the N-terminal region of the winged-helix motif. The arrangement of helix α4 in relation to the winged-hel
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