Portal de Periódicos da CAPES

Tetrameric Architecture of the Circadian Clock Protein KaiB

2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês

10.1074/jbc.m411284200

1083-351X

Kenichi Hitomi, Tokitaka Oyama, Seungil Han, A.S. Arvai, Elizabeth D. Getzoff,

Light effects on plants

Cyanobacteria are among the simplest organisms that show daily rhythmicity. Their circadian rhythms consist of the localization, interaction, and accumulation of various proteins, including KaiA, KaiB, KaiC, and SasA. We have determined the 1.9-Å resolution crystallographic structure of the cyanobacterial KaiB clock protein from Synechocystis sp. PCC6803. This homotetrameric structure reveals a novel KaiB interface for protein-protein interaction; the protruding hydrophobic helix-turn-helix motif of one subunit fits into a groove between two β-strands of the adjacent subunit. A cyanobacterial mutant, in which the Asp-Lys salt bridge mediating this tetramer-forming interaction is disrupted by mutation of Asp to Gly, exhibits severely impaired rhythmicity (a short free-running period; ∼19 h). The KaiB tetramer forms an open square, with positively charged residues around the perimeter. KaiB is localized on the phospholipid-rich membrane and translocates to the cytosol to interact with the other Kai components, KaiA and KaiC. KaiB antagonizes the action of KaiA on KaiC, and shares a sequence-homologous domain with the SasA kinase. Based on our structure, we discuss functional roles for KaiB in the circadian clock. Cyanobacteria are among the simplest organisms that show daily rhythmicity. Their circadian rhythms consist of the localization, interaction, and accumulation of various proteins, including KaiA, KaiB, KaiC, and SasA. We have determined the 1.9-Å resolution crystallographic structure of the cyanobacterial KaiB clock protein from Synechocystis sp. PCC6803. This homotetrameric structure reveals a novel KaiB interface for protein-protein interaction; the protruding hydrophobic helix-turn-helix motif of one subunit fits into a groove between two β-strands of the adjacent subunit. A cyanobacterial mutant, in which the Asp-Lys salt bridge mediating this tetramer-forming interaction is disrupted by mutation of Asp to Gly, exhibits severely impaired rhythmicity (a short free-running period; ∼19 h). The KaiB tetramer forms an open square, with positively charged residues around the perimeter. KaiB is localized on the phospholipid-rich membrane and translocates to the cytosol to interact with the other Kai components, KaiA and KaiC. KaiB antagonizes the action of KaiA on KaiC, and shares a sequence-homologous domain with the SasA kinase. Based on our structure, we discuss functional roles for KaiB in the circadian clock. In bacteria to humans, daily rhythms endogenously consist of ever-changing protein localization, accumulation, and interactions (for reviews of cyanobacterial clocks, see Refs. 1Golden S.S. Ishiura M. Johnson C.H. Kondo T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 327-354Crossref PubMed Scopus (168) Google Scholar, 2Golden S.S. Canales S.R. Nat. Rev. Microbiol. 2003; 1: 191-199Crossref PubMed Scopus (77) Google Scholar, 3Golden S.S. Curr. Opin. Microbiol. 2003; 6: 535-540Crossref PubMed Scopus (25) Google Scholar, 4Ditty J.L. Williams S.B. Golden S.S. Annu. Rev. Genet. 2003; 37: 513-543Crossref PubMed Scopus (97) Google Scholar, 5Johnson C.H. Nature. 2004; 430: 23-24Crossref PubMed Scopus (9) Google Scholar). In cells, proteins involved in the circadian clock interact with other clock components, sometimes self-assemble, and take part in a complex protein network with the right timing, placement, and organization to create and maintain a robust rhythm. Meanwhile, the clock core is adjusted by exogenous factors, e.g. light. In any organism, biological phenomena defined as a circadian clock have three characteristic features: a free running periodicity of about 24 h, phase resetting by environmental cues, and temperature compensation of the period. The proper associations among individual clock proteins are essential, but their interplay with each other can be subtle and difficult to decipher. Cyanobacteria, the evolutionary predecessors of chloroplasts, are both the simplest photosynthetic cells and the most primitive organisms that maintain a circadian clock. The circadian clock gene cluster, kaiABC, was originally discovered and cloned from the cyanobacterium Synechococcus elongatus PCC7942 (6Ishiura 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 (580) Google Scholar). Interestingly, kaiB and kaiC homologues of unknown function have been discovered in Archaea and Proteobacteria, but the three kai genes have very different evolutionary histories (7Dvornyk V. Vinogradova O. Nevo E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2495-2500Crossref PubMed Scopus (176) Google Scholar). In S. elongatus PCC7942, however, inactivation of any single kai gene abolishes the circadian rhythms (6Ishiura 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 (580) Google Scholar). The KaiA, KaiB, and KaiC proteins can associate in all possible combinations, including self-assembly in vitro and in vivo (8Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (140) Google Scholar). Crystal and solution structures of cyanobacterial KaiA domains reveal a dimer with tight interactions (9Williams S.B. Vakonakis I. Golden S.S. LiWang A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15357-15362Crossref PubMed Scopus (172) Google Scholar, 10Uzumaki T. Fujita M. Nakatsu T. Hayashi F. Shibata H. Itoh N. Kato H. Ishiura M. Nat. Struct. Mol. Biol. 2004; 11: 623-631Crossref PubMed Scopus (66) Google Scholar, 11Ye S. Vakonakis I. Ioerger T.R. LiWang A.C. Sacchettini J.C. J. Biol. Chem. 2004; 279: 20511-20518Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Garces R.G. Wu N. Gillon W. Pai E.F. EMBO J. 2004; 23: 1688-1698Crossref PubMed Scopus (71) Google Scholar, 13Vakonakis I. Sun J. Wu T. Holzenburg A. Golden S.S. LiWang A.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1479-1484Crossref PubMed Scopus (55) Google Scholar). In the structure of the full-length KaiA dimer, the two independently folded domains are connected by a canonical linker with domain swapping (11Ye S. Vakonakis I. Ioerger T.R. LiWang A.C. Sacchettini J.C. J. Biol. Chem. 2004; 279: 20511-20518Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The two domains of the KaiC protein, which belongs to the family of ATPase/GTPase homologues, stack into a dumbbell shape and assemble into a symmetric double-layered hexameric ring in an ATP-dependent manner (13Vakonakis I. Sun J. Wu T. Holzenburg A. Golden S.S. LiWang A.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1479-1484Crossref PubMed Scopus (55) Google Scholar, 14Hayashi F. Suzuki H. Iwase R. Uzumaki T. Miyake A. Shen J.R. Imada K. Furukawa Y. Yonekura K. Namba K. Ishiura M. Genes Cells. 2003; 8: 287-296Crossref PubMed Scopus (109) Google Scholar, 15Pattanayek R. Wang J. Mori T. Xu Y. Johnson C.H. Egli M. Mol. Cell. 2004; 15: 375-388Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The histidine kinase designated SasA closely associates with the cyanobacterial clock machinery, and is necessary for maintaining robust circadian rhythms, although the SasA gene lies outside the kaiABC cluster (16Iwasaki H. Williams S.B. Kitayama Y. Ishiura M. Golden S.S. Kondo T. Cell. 2000; 101: 223-233Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Interestingly, SasA contains a KaiB-like sensory domain that mediates binding of SasA to KaiC. The Kai and SasA proteins dynamically associate into heteromultimeric protein complexes in a circadian fashion (19Kageyama H. Kondo T. Iwasaki H. J. Bio. Chem. 2003; 278: 2388-2395Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). KaiA enhances KaiC phosphorylation and KaiB attenuates KaiA-enhanced KaiC autokinase activity (17Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (230) Google Scholar, 18Xu Y. Mori T. Johnson C.H. EMBO J. 2003; 22: 2117-2126Crossref PubMed Scopus (190) Google Scholar). The KaiB function as an attenuator of KaiC phosphorylation requires the presence of KaiA, suggesting that a heteromultimeric KaiABC protein complex would be formed following the KaiA-KaiC interaction. In in vitro studies, the KaiA dimer interaction with the KaiC hexamer was suggested to vary from 1:1 (two molecules of KaiA against six molecules of KaiC) to up to 2:1, depending on ATP hydrolysis by KaiC. It is still unknown how KaiB stoichiometrically interacts with the KaiA-KaiC complex. Recently, a crystal structure of the Anabaena KaiB homologue was determined, showing that the KaiB homologue is a dimer in the crystal as are the KaiA structures (12Garces R.G. Wu N. Gillon W. Pai E.F. EMBO J. 2004; 23: 1688-1698Crossref PubMed Scopus (71) Google Scholar). On the other hand, it was shown in vivo that the KaiC-based complex changes in size in response to day and night, accompanied by the night-specific interaction with KaiA and KaiB (19Kageyama H. Kondo T. Iwasaki H. J. Bio. Chem. 2003; 278: 2388-2395Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In the cyanobacterial cell, although the amount of KaiA is constant, KaiB and KaiC exhibit robust circadian rhythms, being the most abundant at the early circadian night phase (circadian time 15–16). The amount of KaiB and KaiC changes rhythmically with the KaiB:KaiC ratio up to 2:1 (17Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (230) Google Scholar), even though these two genes form an operon (6Ishiura 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 (580) Google Scholar). Kitayama et al. (17Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (230) Google Scholar) showed that KaiB protein is localized in both the cytosol and the membrane, suggesting that a KaiB regulatory link between subcellular localization and protein-protein interactions is important in the cyanobacterial clock system. The KaiB protein is essential to the circadian rhythm of cyanobacteria: rhythmicity disappearing in the kaiB null mutant (6Ishiura 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 (580) Google Scholar). Because most kaiB mutants showed a short period phenotype (6Ishiura 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 (580) Google Scholar), hypomorph mutations are likely to shorten the period. Although genetic evidence shows a key role for KaiB in pacemaking, the biochemistry of KaiB function is poorly understood. Here we report the crystallographic structure of the KaiB tetramer from Synechocystis sp. PCC6803, identify interesting structural features, including the (i) positively charged perimeter, (ii) negatively charged center, and (iii) zipper of aromatic rings, and discuss their implications for KaiB function. Furthermore, we demonstrate that a mutation at the tetrameric interface affects the clock, suggesting that the inability to form this newly identified tetrameric complex may cause abnormality in the circadian rhythm. Expression and Purification of Synechocystis sp. PCC6803 KaiB— The Synechocystis sp. PCC6803 kaiB gene was introduced into a pET11a (Novagen) vector. Escherichia coli BL21(DE3) cells transformed by this plasmid were grown at 30 °C and protein expression was induced with 0.2 mm isopropyl β-d-thiogalactopyranoside. The cells were disrupted by sonication, and the supernatant was applied to anion exchange (Poros 20 HQ), cation exchange (Poros HS), and gel filtration (S-200 26/60) columns. The anion and cation exchange columns did not bind the cyanobacterial protein, whereas the other E. coli-derived proteins were efficiently removed. The protein yield was 30 mg/liter. Purified KaiB protein was concentrated by Amicon Centriprep (Millipore) to 10 mm in a solution containing 20 mm Tris-HCl (pH 7.5), 200 mm NaCl, and 0.5 mm EDTA, and stored at -80 °C. Dynamic Light Scattering—Dynamic light scattering studies were performed at 20 °C by using a DynaPro 99 instrument (Protein Solutions, Inc.). Scattering of Synechocystis sp. PCC6803 KaiB was analyzed at 10 mm in the storage buffer (20 mm Tris-HCl (pH 7.5), 200 mm NaCl, and 0.5 mm EDTA). The DynaPro 99 instrument measures fluctuations in the intensity of scattered laser light caused by the Brownian motion of molecules in solution. Reported scattering values are the averages of 30 scans, each including measurements at 20 different time points between 3 and 3000 μs. Data were analyzed with DYNAMICS version 5.5.56.38 software (Protein Solutions, Inc.). The scattering data are fitted with an exponential autocorrelation function, which is used to determine the molecular translational diffusion coefficient, DT, and to assess the polydispersity of the sample (Table I). The radius of hydration, RH, is then calculated by using the equation DT = kT/6πηRH, where k is the Boltzmann constant, T is temperature in Kelvin, and η is the solvent viscosity. RH is defined as the radius of a hypothetical hard sphere that diffuses with the same speed as the particle under examination. But macromolecules are non-spherical and solvated. Therefore, the molecular weight (Mr) of a macromolecule is estimated by using Mrversus RH calibration curves developed from standards of known molecular weight and size. Thus, the Mr estimate of a given particle is subject to error if it deviates from the shape and solvation of the molecules used as standards. The molecular weight for protein macromolecules is estimated from a curve that fits the following Mr = [1.6800·RH]2.3398, as implemented in the software. Statistics for dynamic light scattering are summarized in Table I.Table IDynamic light scattering study of Synechocystis sp. PCC6803 KaiBDTaDT, translational diffusion coefficientRHbRH, hydrodynamic radiusMassPolydispersity10–9cm2/snmkDa%646.33.25153.29.2a DT, translational diffusion coefficientb RH, hydrodynamic radius Open table in a new tab Crystallization and Diffraction Data Collection—Crystals of the Synechocystis sp. PCC6803 KaiB protein were obtained at both 4 °C and room temperature, by the hanging drop method. The 2-microliter drop, containing a 1:1 ratio (v/v) of protein (10 mm in storage buffer) to reservoir solution (below) was equilibrated against 500 ml of a reservoir solution containing 60–80% saturated betaine and 100 mm imidazole/malate buffer (pH 6.0). Crystals grown at 4 °C diffracted to higher resolution than those grown at room temperature, giving diffraction data to 1.9-Å resolution data at SSRL Beam Line 7-1. The data were processed by DENZO/SCALEPACK (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). KaiB crystals belong to space group P212121 with unit cell dimensions: a = 52.8 Å, b = 66.0 Å, c = 114.8 Å. Four monomers per asymmetric unit give a 41.3% solvent content. Model Building and Refinement—Phases were obtained by molecular replacement with the AMoRE program package (21Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar). Results with the Anabaena KaiB dimer (Protein Data Bank code 1R5P) search model gave a correlation factor of 54.3% and an R factor of 41.1% in resolution range from 10 to 4 Å. Structure refinement was carried out with CNS (22Brünger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar), with iterations of positional refinement, simulated annealing, and torsion angle dynamics followed by temperature factor (B factor) refinement. The model fitting was done with Xfit (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). Statistics for crystallographic data processing and refinement are summarized in Table II. The coordinates for Synechocycystis KaiB are deposited in the RCSB Protein Data Bank under the Protein Data Bank code 1WWJ.Table IISynechocystis sp. KaiBDiffraction dataBeamlineSSRL 7-1Resolution (Å)30–1.9 (1.97-1.90)aValues for the highest resolution shell are given in parenthesesWavelength (Å)1.08Observations96,330Unique reflection31,143 (2,882)Completeness (%)95.9 (89.9)RsymbRsym = the unweighted R value on I between symmetry mates3.7 (25.1)I/σ(I)cI is the observed intensity and σ(I) is the standard deviation of I23.9 (3.9)Space groupP212121a = 52.8b = 66.0c = 114.8Molecular in asymmetric units4Refinements statisticsReflections29,369R factordR factor = Σhkl||Fobs|–k|Fcalc||/Σhkl|Fobs|0.249RfreeeRfree is the cross-validation R factor for 5% of reflections against which the model was not refined0.283r.m.s.dfr.m.s.d., root mean square deviation bond lengths0.0065r.m.s.d bond angles1.369a Values for the highest resolution shell are given in parenthesesb Rsym = the unweighted R value on I between symmetry matesc I is the observed intensity and σ(I) is the standard deviation of Id R factor = Σhkl||Fobs|–k|Fcalc||/Σhkl|Fobs|e Rfree is the cross-validation R factor for 5% of reflections against which the model was not refinedf r.m.s.d., root mean square deviation Open table in a new tab Mutational Analysis: Measurement of Circadian Rhythms of Bioluminescence in S. elongatus PCC7942—Bioluminescence rhythm assays used cyanobacterial strains, culture conditions, and methods described previously (24Nishimura H. Nakahira Y. Imai K. Tsuruhara A. Kondo H. Hayashi H. Hirai M. Saito H. Kondo T. Microbiology. 2002; 148: 2903-2909Crossref PubMed Scopus (46) Google Scholar). NUC42 and NUC43 were used as wild type and ΔkaiABC deletion strains, respectively. The S. elongatus PCC7942 kaiB8 (D90G) mutant was originally isolated from a PCR-based random mutagenesis library at the kaiB locus. 1T. Oyama, Y. Nakahira, S. Takeuchi, and T. Kondo, unpublished data. The library was constructed by using the strategy described for kaiA mutagenesis by Nishimura et al. (24Nishimura H. Nakahira Y. Imai K. Tsuruhara A. Kondo H. Hayashi H. Hirai M. Saito H. Kondo T. Microbiology. 2002; 148: 2903-2909Crossref PubMed Scopus (46) Google Scholar), except for the restriction enzymes (AflII and HpaI) to target the kaiB locus. Because the original mutant strain was gone, we recreated the mutant construct by PCR-based site-directed mutagenesis. The PCR product was amplified by using the primer set (5′-AACGACAGTTAGAAGTCGTCGGAATCTTGAAGTTCGCCGTAGAGTAAACCAAGGC-3′ (55-mer) and 5′-GAGGACATTTTGCTGGATTA-3′ (20-mer)), digested with AflII and HpaI and introduced into the pCkaiABC vector from which the kaiB locus had been excised using AflII and HpaI (24Nishimura H. Nakahira Y. Imai K. Tsuruhara A. Kondo H. Hayashi H. Hirai M. Saito H. Kondo T. Microbiology. 2002; 148: 2903-2909Crossref PubMed Scopus (46) Google Scholar). Overall Tetrameric KaiB Structure—We obtained our best diffraction quality crystals of Synechocystis KaiB by using trimethylglycine, also known as betaine, as a precipitant. Synechocystis KaiB also crystallized as thin plates from PEG 400 under conditions resembling those used for crystallization of Anabaena KaiB (12Garces R.G. Wu N. Gillon W. Pai E.F. EMBO J. 2004; 23: 1688-1698Crossref PubMed Scopus (71) Google Scholar), but these crystals diffracted poorly. The KaiB crystals grown from betaine belong to space group P212121 with unit cell dimensions a = 52.8 Å, b = 66.0 Å, c = 114.8 Å, and four 105-residue KaiB polypeptide chains per asymmetric unit, giving a 41% solvent content (Table II). The four subunits (two dimers) in each asymmetric unit of the crystal compose half of two tetramers. Dynamic light scattering experiments (Fig. 1 and Table I) also indicated that Synechocystis KaiB (11.9 kDa) in aqueous solution is predominantly (∼90%) in a single assembly state (polydispersion ∼10%), corresponding to a tetramer (hydrodynamic radius of 3.25, apparent molecular weight of 53.2). In the crystal, pairs of KaiB dimers related by non-crystallographic symmetry interact to form a flattened tetramer (dimensions of 48 × 67 × 28 Å) (Fig. 2, A and B). Pairs of anti-parallel α-helices form each of the four sides of a square box, which resembles the Japanese character "Kai," meaning cycle. This box is filled with four β-sheets and the top and bottom are each half-covered by two pairs of α-helices.Fig. 2Overall structure of tetrameric Synechocystis sp. PCC6803 KaiB. A, Synechocystis KaiB, shown as a ribbon diagram, forms flattened (side view) and Kai-like (top view) structure: α-helices, green; β-strands, yellow; coil, pink. B, tetrameric Synechocystis KaiB assembles from two symmetric homodimers. We define the dimer interface as Interface I, and the interface between dimers as Interface II. C, electrostatic potential surface for tetrameric KaiB complex reveals characteristic features: a positively charged rim (side view) and a negatively charged cluster at the center of one Kai face (top and bottom views). Electrostatic potential color code is from red, -2.0 kT/e, to blue, +2.0 kT/e.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The electrostatic potential surface of tetrameric KaiB reveals a pattern of positive charges with functional implications (Fig. 2C). Positively charged residues are located around the perimeter of the box: Lys6, Arg23, Lys26, Lys29, Lys49, Arg74, Lys75, Arg83, and Lys85. Eight glutamates, Glu55, Glu56, Asp95, and Glu96 of two facing molecules, make a negatively charged cluster at the center of the tetramer. Along the diagonal, four aromatic residues, Tyr8, Phe36, Tyr40, and Tyr94, line the interface between dimers (Fig. 3, A and B). At the end of these aromatic zipper residues, Tyr94 packs against a cluster of three consecutive prolines (Pro70–Pro71–Pro72), forming additional hydrophobic interactions that connect the tetramer (see Fig. 5B).Fig. 5Synechocystis KaiB assembly interfaces. A, the dimer interface (Interface I) is meditated by main chain hydrogen bonds joining symmetry related Ile59 residues to form a anti-parallel β sheet from β3 of each subunit: molecule 3 main chain, pale yellow; molecule 3 side chains, green; molecule 4 main chain, purple; molecule 4, side chains, yellow. B, Interface II, between dimers within the tetramer, is made by fitting a helix-turn-helix motif into the groove between β3 and β4 of the adjacent subunit: molecule 1 main chain, red; molecule 1 side chains, green; molecule 4 main chain, purple; molecule 4 side chains, yellow. C and D, comparison between Synechocystis KaiB (red) and Anabaena KaiB (yellow) in the tetrameric structure complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also found five trimethylglycine (betaine) molecules bound to the KaiB tetramer, primarily through interactions of the trimethyl moiety with electronegative groups on the protein. Pairs of betaine molecules bind in hydrophilic environments at the interface between KaiB dimers near Gln34–Glu35–(Phe36)–Gln37 at diagonally opposed corners of the box-shaped KaiB tetramer (Fig. 3C). One trimethylglycine was uniquely bound at the end of the aromatic zipper in a negatively charged pocket between two KaiB dimers (Fig. 3, D and E). This negative pocket is surrounded with positive residues. KaiB Subunit Fold in the Tetramer—Each Synechocystis sp. PCC6803 KaiB subunit in the tetramer consists of three α helices and four β strands in the shape of a wedge (Fig. 4). The three longest β strands form a mixed β-sheet with N-terminal β1 in the center. β2 is parallel to β1, and β4 is anti-parallel to β1 and β2 (Fig. 4A). The two longest α helices (α1 and α3), which precede and are anti-parallel to β2 and β4, respectively, shield one side of this β-sheet. The shortest β strand (β3) hydrogen bonds with its symmetry mate across the dimer interface to form a small anti-parallel β-sheet, roughly perpendicular to the larger three-stranded β-sheet (Fig. 5A). Except for the five N-terminal and 10 C-terminal amino acids, the Synechocystis KaiB subunit is structurally well conserved with Anabaena KaiB (root mean square deviation of 1.27 Å for Cα) (Fig. 4B). This suggests that the core domain of KaiB homologues is structurally conserved throughout cyanobacteria (7Dvornyk V. Vinogradova O. Nevo E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2495-2500Crossref PubMed Scopus (176) Google Scholar). In the Synechocystis KaiB tetrameric structure, the N termini have predominantly well ordered electron density (Fig. 4C). Based upon their location within the box-like tetramer (Fig. 2, A and B), the N termini are classified into two groups: inner and outer (Fig. 4D). The inner N termini protrude from the center of the Kai box (Fig. 2A), whereas the outer N termini hold the adjacent dimer, strengthening the tetrameric structure (Fig. 2B). KaiB Oligomeric Interfaces—The tetrameric KaiB structure (Fig. 2B) reveals two different interfaces for KaiB (Fig. 5). Interface I exhibits non-crystallographic 2-fold symmetry (Fig. 5A) and is closely related to the dimer interface in the Anabaena KaiB dimer structure (12Garces R.G. Wu N. Gillon W. Pai E.F. EMBO J. 2004; 23: 1688-1698Crossref PubMed Scopus (71) Google Scholar). In the Synechocystis complex structure, this dimer interface is centered on the main chain, antiparallel, β-strand hydrogen bonds between Ile59 residues of β3 in each subunit, and is mediated by a loop from Leu48 to Ala61, encompassing β3. The Gln52 side chain makes a hydrogen bond with the backbone oxygen of Leu87. The backbone nitrogen of Leu53 makes a hydrogen bond with the Ile88 backbone oxygen. The carbonyl oxygen atoms of Glu55 and Asp57 are linked through a water molecule to the nitrogen of Ala61. In addition to these hydrophilic interactions, several hydrophobic interactions occur between Leu53 and Ile88, Leu53, and Ile59, and Ile59 and Ile59. Overall each subunit of the dimer buries almost 800 Å2 of surface area in Interface I. To assemble dimers into the KaiB tetramer, the helix-turn-helix motif between α2 and α3 (Thr62–Asp82) fits into a cleft between β3 and β4 (Interface II, Fig. 5B). Pro70 and Pro72 are in van der Waals contact with Phe36 and Tyr94, respectively. The Lys67 and Glu55 side chains form a salt bridge, and the Ile68 backbone oxygen interacts with Nζ of Lys58. Adding to these interactions between dimers, the Tyr40 hydroxyl forms hydrogen bonds with the backbone carbonyl oxygen of Leu92 and Nζ of Lys58 through a water molecule. Residues Phe36, Tyr40, Tyr8, and Tyr94 from all four subunits form a zipper of aromatic residues across this tetramer interface (Fig. 3, A and B). At the edges of Interface II, the "outer" N termini stabilize the assembly of two KaiB dimers into the tetramer. The N terminus extends toward the outside of the Kai box and hooks to the adjacent dimer (Fig. 2, A and B). The Tyr8 hydroxyl makes a hydrogen bond with Glu35, and in one tetramer the Ser2 backbone oxygen and nitrogen make hydrogen bonds with the side chain of Gln34 (Fig. 5B). Overall, each dimer buries more than 1900 Å2 of surface area in Interface II to form the tetramer. Mutation at the Tetrameric Interface Disrupts the Circadian Clock—We identified an interesting kaiB mutant, with a substitution in the tetramer interface. This Asp → Gly mutant (position 90 in Synechococcus equivalent to position 91 in Synechocystis) exhibits arrhythmicity. 2T. Oyama, Y. Nakahira, S. Takeuchi, and T. Kondo, unpublished data. In the tetrameric Synechocystis structure, a salt bridge between Asp91 and Lys58 orients Lys to form hydrogen bonds across dimer-dimer Interface II to Tyr40 and the backbone oxygen of Ile68. By precluding this salt bridge and leaving an unsatisfied positive charge, the D90G substitution likely weakens or disrupts KaiB tetrameric assembly. To test the resultant phenotypic effect on circadian rhythm, we used the bioluminescence rhythm assay to monitor the mutant S. elongatus cells (Fig. 6). The parental wild type strain, which carries a luciferase reporter gene under control of the kaiBC promoter, exhibits normal circadian rhythmicity (Fig. 6A), whereas the kaiABC deletion mutant of this strain was arrhythmic (Fig. 6B, Ref. 5Johnson C.H. Nature. 2004; 430: 23-24Crossref PubMed Scopus (9) Google Scholar). Reintroduction of the wild type kaiABC locus restored normal rhythmicity (Fig. 6C), whereas introduction of the kaiABC locus carrying the D90G mutation in kaiB showed a severely impaired rhythmicity (a short free-running period; 19 h), which damped to arrythmicity in 3 days under continuous light conditions (Fig. 6D). These results suggest that disruption of the KaiB tetramer severely diminishes the molecular function of KaiB. Structural Analysis of the KaiB Monomer—The structures of KaiABC proteins provide the basis for mutational mapping to help decipher protein function and protein-protein interactions (9Williams S.B. Vakonakis I. Golden S.S. LiWang A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15357-15362Crossref PubMed Scopus (172) Google Scholar, 10Uzumaki T. Fujita M. Nakatsu T. Hayashi F. Shibata H. Itoh N. Kato H. Ishiura M. Nat. Struct. Mol.