Structural model of the circadian clock KaiB–KaiC complex and mechanism for modulation of KaiC phosphorylation
2008; Springer Nature; Volume: 27; Issue: 12 Linguagem: Inglês
10.1038/emboj.2008.104
ISSN1460-2075
AutoresRekha Pattanayek, Dewight Williams, Sabuj Pattanayek, Tetsuya Mori, Carl Hirschie Johnson, Phoebe L. Stewart, Martin Egli,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle22 May 2008free access Structural model of the circadian clock KaiB–KaiC complex and mechanism for modulation of KaiC phosphorylation Rekha Pattanayek Rekha Pattanayek Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Dewight R Williams Dewight R Williams Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Sabuj Pattanayek Sabuj Pattanayek Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Tetsuya Mori Tetsuya Mori Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Carl H Johnson Carl H Johnson Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Phoebe L Stewart Phoebe L Stewart Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Martin Egli Corresponding Author Martin Egli Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Rekha Pattanayek Rekha Pattanayek Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Dewight R Williams Dewight R Williams Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Sabuj Pattanayek Sabuj Pattanayek Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Tetsuya Mori Tetsuya Mori Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Carl H Johnson Carl H Johnson Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Phoebe L Stewart Phoebe L Stewart Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Martin Egli Corresponding Author Martin Egli Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Author Information Rekha Pattanayek1,‡, Dewight R Williams2,‡, Sabuj Pattanayek1, Tetsuya Mori3, Carl H Johnson3, Phoebe L Stewart2 and Martin Egli 1 1Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA 2Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA 3Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA ‡These authors contributed equally to this work *Corresponding author. Department of Biochemistry, School of Medicine, Vanderbilt University, 607 Light Hall, Nashville, TN 37232, USA. Tel.: +1 615 343 8070; Fax: +1 615 322 7122; E-mail: [email protected] The EMBO Journal (2008)27:1767-1778https://doi.org/10.1038/emboj.2008.104 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB–KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA. Introduction The circadian clock of the cyanobacterial model organism Synechococcus elongatus can be reconstituted in vitro from the three proteins KaiA, KaiB and KaiC in the presence of ATP and Mg2+ (Nakajima et al, 2005). The in vitro system exhibits the two main characteristics of circadian clocks (Dunlap et al, 2004), a regular rhythm with a period of ca. 24 h and temperature compensation. The KaiC protein constitutes the central cog of the clock and forms a hexamer (Mori et al, 2002; Hayashi et al, 2003; Pattanayek et al, 2004) with auto-kinase and auto-phosphatase activity in vitro (Xu et al, 2003; Nishiwaki et al, 2004). KaiC rhythmically oscillates between hypo- and hyper-phosphorylated forms in vivo (Xu et al, 2003; Nishiwaki et al, 2004), and its phosphorylation status is correlated with the period of the clock (Xu et al, 2003). KaiA enhances KaiC phosphorylation and KaiB exert an effect as a antagonist of KaiA (Iwasaki et al, 2002; Williams et al, 2002, Kitayama et al, 2003; Xu et al, 2003; Nishiwaki et al, 2004). The discoveries that the KaiABC clock ticks in the absence of a transcription–translation oscillatory feedback loop (Tomita et al, 2005), and that it can be reconstituted from three proteins (Nakajima et al, 2005) render this molecular timer a unique target for detailed biochemical and biophysical investigations. In particular, a better understanding of the interactions among the three Kai proteins will be key to gaining insight into the ability of this relatively simple system to sustain a robust oscillation between phosphorylated and dephosphorylated states with a 24-h period. Three-dimensional (3D) structures for the KaiA (Williams et al, 2002; Garces et al, 2004; Uzumaki et al, 2004; Vakonakis and LiWang, 2004; Ye et al, 2004), KaiB (Garces et al, 2004; Hitomi et al, 2005; Iwase et al, 2005) and KaiC (Pattanayek et al, 2004) proteins or domains thereof (KaiA) have been determined by X-ray crystallography (KaiA, KaiB and KaiC) or NMR (KaiA). S. elongatus KaiC (SeKaiC) takes the shape of a double doughnut with 12 ATP-binding sites between N-terminal KaiCI and C-terminal KaiCII domains. Interactions between KaiC subunits and ATP in the CI half involve hydrogen bonds to the nucleobase portion that are absent in the CII half (Pattanayek et al, 2004), consistent with a tighter binding of ATP molecules in the CI compared with the CII ring (Hayashi et al, 2004a). Crystallographic data allowed identification of phosphorylation sites in the CII half, T432 and S431, and the phosphorylated form of KaiC (P-KaiC) exhibits more extensive subunit interactions relative to the non-phosphorylated form (Xu et al, 2004). Interestingly, the dephosphorylation phase of the KaiC hexamer triggered by KaiB binding is accompanied by KaiC subunit exchange (Kageyama et al, 2006), a process that appears to synchronize the phosphorylation state of KaiC particles and is important for sustaining a high-amplitude oscillation (Mori et al, 2007). The S. elongatus KaiA (SeKaiA) protein forms a domain-swapped dimer, with the C-terminal dimerization and KaiC-interacting domain adopting a four-helix bundle fold (Ye et al, 2004). The latter finding was confirmed by structures of the C-terminal domain of KaiA from Anabaena sp. PCC7120 (Garces et al, 2004) and KaiA from Thermosynechococcus elongatus BP-1 (ThKaiA) (Uzumaki et al, 2004; Vakonakis et al, 2004). In the NMR structure of a complex between the ThKaiA C-terminal domain and a 30mer C-terminal peptide from ThKaiC two peptides are bound inside grooves above the dimerization interface on opposite faces of the KaiA dimer (Vakonakis and LiWang, 2004). However, this observation did not provide an explanation for KaiA's ability to enhance KaiC phosphorylation. On the basis of single-particle EM reconstruction of the negatively stained T. elongatus BP-1 KaiA–KaiC (ThKaiAC) complex and in vitro studies of complex formation between wild-type and mutant KaiA and KaiC proteins, we recently derived a model for the 3D structure of a KaiAC complex with 1:1 stoichiometry (one KaiA dimer:one KaiC hexamer) (Pattanayek et al, 2006). Tethering the KaiA dimer to the KaiC hexamer through the flexible C-terminal peptide from a KaiC subunit potentially allows for a second more transitory interaction between an apical loop from a KaiA monomer and a KaiCII ATP-binding cleft. The KaiAC model involved extending the KaiC residues in the S-shaped loop (aa 485–497) of the KaiC crystal structure to form a flexible linker between the KaiA dimer and the hexameric barrel of KaiC. The model is consistent with all of the structural information of the component proteins (Egli et al, 2007), the observation that a single KaiA dimer is able to interact with the KaiC hexamer and enhance the latter's auto-kinase activity (Hayashi et al, 2004b), and rapid and repeated action of KaiA on KaiC during the phosphorylation phase for up to 6 h after start of incubation (Kageyama et al, 2006). Little is known at the moment regarding the mechanism of KaiB-induced dephosphorylation of the KaiC hexamer and the binding mode between KaiB and KaiC. Crystal structures of KaiB from Anabaena sp. PCC7120 (Garces et al, 2004), Synechocystis PCC6803 (Hitomi et al, 2005) and T. elongatus BP-1 (T64C–ThKaiB mutant) (Iwase et al, 2005) revealed a thioredoxin-like fold of the monomer. In the crystals and in solution, KaiB forms a tetramer with a positively charged perimeter, a negatively charged centre and a zipper of aromatic rings important for oligomerization (Hitomi et al, 2005). On the basis of altered rhythm caused by mutant KaiBs in vivo, it was concluded that the tetrameric state is important for proper clock function (Hitomi et al, 2005). However, a recent in vitro analysis of Kai protein complexes during the KaiC phosphorylation cycle relying on gel filtration chromatography found that KaiB appeared to bind to KaiC as a dimer (Kageyama et al, 2006). Interestingly, association and dissociation between KaiB and KaiC was slower compared with the KaiA–KaiC interaction, with only about 5–20% of KaiCs bound to KaiB. KaiA binds to non-phosphorylated KaiC, the phosphorylated form of KaiC (P-KaiC), with or without KaiB bound, as well as to a phosphorylation-site double mutant (S431A/T432A; Kageyama et al, 2006). By contrast, KaiB associates most easily with the P-KaiC form. In addition, KaiA does interfere with KaiC subunit exchange but KaiB promotes it. It is clear that KaiB action does not lead to a dissociation of KaiA from KaiC; rather the data are consistent with the existence of a ternary KaiABC complex during the dephosphorylation phase (Kageyama et al, 2006; Mori et al, 2007). Using a combination of approaches, including X-ray crystallography, negative-stain and cryo-electron microscopy (EM), gel electrophoresis, mutagenesis and modelling techniques, we have built a 3D model of the S. elongatus KaiB–KaiC (SeKaiBC) complex. The experimental data are consistent with the binding of two KaiB dimers on the C-terminal dome-shaped surface presented by the KaiCII hexamer. The 3D model of this complex together with that of the ThKaiAC complex (Pattanayek et al, 2006) readily suggests mechanisms by which KaiB antagonizes KaiA-stimulated enhancement of KaiC phosphorylation. It also suggests a possible structural explanation for the critical role played by the C-terminal region of KaiB in the KaiC dephosphorylation phase (Iwase et al, 2005). The KaiBC model also explains the observation of simultaneous binding of KaiA and KaiB to KaiC and leads to the idea that all three proteins interact through their C-terminal regions. Results X-ray crystal structure of wild-type KaiB from T. elongatus BP-1 The crystal structure of the full-length wild-type KaiB protein (M1-E108) from T. elongatus BP-1 (ThKaiB) was determined at 2.7 Å by the molecular replacement method, using the previously published structure of the T64C ThKaiB mutant as a model (Iwase et al, 2005). Selected crystal data and refinement statistics are summarized in Table I and stereo diagram illustrating the quality of the final electron density is depicted in Figure 1. Final electron densities around individual N- and C-terminal tails are depicted in Supplementary Figure S1. The structure in space group P21212 reveals six KaiB molecules, termed A–F, per crystallographic asymmetric unit (a.u.; Figure 1A). These form two tetramers of which one is positioned on a crystallographic dyad (Figure 1C). Structures of KaiB tetramers had previously been determined for the proteins from Anabaena (one dimer per a.u.; Garces et al, 2004), Synechocystis (two dimers per a.u.; Hitomi et al, 2005) and a T64C T. elongatus mutant (two dimers per a.u.; Iwase et al, 2005). However, no structures have been reported for the wild-type SeKaiB or ThKaiB (this work) proteins to date. The wild-type ThKaiB structure is the first to include the C-terminal tail, which is either extended or folded under the rest of the KaiB monomer. The core residues (aa 8–94) of the six ThKaiB monomers in the asymmetric unit show close resemblance to each other and to the KaiB structures determined earlier. The r.m.s. deviations for residues 8–94 (87 atom pairs) among independent ThKaiB molecules are 0.52 (A:C), 0.60 (B:C), 0.65 (D:C), 0.56 (E:C) and 0.63 Å (F:C). The r.m.s. deviations between the C monomer of ThKaiB and monomers in the ThKaiB T64C mutant structure are 0.66 and 0.60 Å. The structure confirms the tetramer as the preferred quaternary structural form of KaiB—at least in the absence of other Kai proteins—in a crystalline form. Preference for a tetrameric assembly in the solution state is indicated by dynamic light scattering (Hitomi et al, 2005), gel filtration, chemical crosslinking and analytical ultracentrifugation (Iwase et al, 2005). Figure 1.Crystal structure of wild-type ThKaiB. (A) The six molecules per crystallographic asymmetric unit illustrating the environments of C-terminal tails in the orthorhombic lattice. The secondary structures, three alpha helices and five beta strands are labelled. Molecules A, B, E and F form a tetramer in a general position and C and D form a second one that is located on a crystallographic dyad. N- and C-terminal residues of the current model are indicated. (B) Stereo diagram illustrating the quality of the omit (2Fo−Fc, 1σ threshold, blue) and Fourier sum electron density (2Fo−Fc, 1σ threshold, orange) in the C-terminal region of the D chain of the ThKaiB crystal structure. (C) The ThKaiB tetramer (molecules C, C′, D and D′) viewed approximately along the crystallographic dyad (indicated by a black dot). Subunits are coloured grey (labelled C and C′) and green (labelled D and D′) and N- and C-terminal residues are labelled. Dimer interfaces (Hitomi et al, 2005) are indicated in Roman numerals and the distance between a pair of Arg residues (R23) across the C/D dimer I interface that matches a similar pair seen in the KaiA dimer (Garces et al, 2004) is shown with a thin line. In addition to the C-terminal tails that contain D and E residues crucial for proper rhythm, side chains of mutants with interesting phenotypes (Ishiura et al, 1998; Hitomi et al, 2005; Iwase et al, 2005) or altered interaction to KaiC (Garces et al, 2004) are highlighted and labelled in one dimer, using the amino-acid numbering previously described for ThKaiB by Iwase et al (2005): K11A (irregular rhythm); L12F (short period, 21 h); R23A (substantially reduced affinity for KaiC); K43A (irregular rhythm); K58A (irregular rhythm); R75F (short period, 21 h); D91G (arrhythmic; mutation disrupts salt bridge between D91 and K58 to orient the latter for a H-bond to Y40 across the dimer II interface). (D) Superimposition of eight ThKaiB molecules based on the crystal structures of the T64C mutant (two molecules, Iwase 2005) and wild-type proteins (six molecules, this work). Residues 8–94 are shown in a single conformation and C-terminal tails beginning with residue 95 are coloured individually, and C-terminal residues are labelled. The colours of individual C-terminal tails match those of molecules in (A). Three of the six C-terminal tails in the crystal structure of wt-ThKaiB were built completely. (E) Electrostatic surface potential of a full-length ThKaiB molecule (molecule D, top panels) and with residues E95–E108 removed (bottom panels) to illustrate the drastic change in the ESP as a result of a lacking C-terminal tail. Electronegative regions are shown in red, neutral regions in white and electropositive regions in blue. (F) Example of the quality of the final electron density around the C terminus of the KaiB D subunit. The stereo diagram shows Fourier sum electron density (2Fo−Fc, 1σ threshold (orange), 0.7σ threshold (grey)) around residue E108 and its neighbouring residues in the crystal lattice. For additional electron density maps and lattice interactions of N- and C-terminal regions see Supplementary Figure S1 and Supplementary Table S1. Download figure Download PowerPoint Table 1. Selected crystal and refinement data for ThKaiBa Data collection Space group P21212 Cell dimensions a, b, c (Å) 100.13, 191.22, 34.34 Wavelength (Å) 1.08 Resolution (last shell; Å) 50–2.78 (2.89–2.78) Unique reflections 16 993 (1797) Completeness (%) 98.5 (97.3) R-merge 0.047 (0.239) I/σ(I) 30.3 (5.3) Refinement Working set reflections (F⩾2σF) 13 727 Test set reflections (F⩾2σF) 1520 Protein atoms 4808 Solvent atoms 63 R-work/R-free (%) 0.233/0.284 Average B-factors (Å2) Protein (all residues) 98 Protein (core residues 8–94) 93 Solvent 99 r.m.s. deviations Bond lengths (Å) 0.008 Bond angles (deg) 1.5 Ramachandran analysis (%) Most favoured 71.4 Allowed 25.6 Generously allowed 3.0 a Values in parentheses refer to the last resolution shell. The C-terminal tail of KaiB was recently shown to be crucial for the function of the protein. In vivo experiments using the S. elongatus PCC 7942 strain demonstrated that a C-terminal deletion comprising residues E95 to F102 resulted in loss of rhythm (Iwase et al, 2005). Similarly, the E95Q/D98N/D100N/D101N SeKaiB quadruple mutant abolished rhythmicity, indicating that the presence of several acidic residues in the C-terminal tail, which is a hallmark of all KaiB proteins, is absolutely required for proper function (for sequence alignments, see Hitomi et al, 2005; Iwase et al, 2005). The availability of multiple independent molecules in a single structure offers the possibility to examine the conformational flexibility of the C-terminal tail. The superimposition reveals considerable deviations between individual C termini (Figure 1D). In the majority of ThKaiB monomers, the C-terminal tails display a curved conformation and fold back such that they lie near the surface of the thioredoxin-fold adopted by the core residues (Figures 1D and E). The individual conformations are undoubtedly influenced by nearest neighbour interactions in the crystal. However, the structure reveals the wide range of conformations that are accessible to the C-terminal tail. Interestingly, in all cases the negatively charged C-terminal tail forms salt bridges with Arg or Lys residues from the same molecule and/or one or more symmetry mates, similar to those shown in Figure 1F for one of the ThKaiB molecules. A full account of the interactions involving acidic residues of KaiB C-terminal tails is provided in Supplementary Table S1. Judging from the conformations of the C termini and the electrostatic properties of their environments in the crystal, we conclude that the tail region is malleable and likely to be involved in electrostatic interactions with partner proteins including KaiC. Furthermore, it is noteworthy that the tails of KaiB proteins from thermophilic strains are typically longer and contain more acidic residues than those from mesophiles. For example, the ThKaiB tail (E95–E108) features three Asp and four Glu residues, whereas the SeKaiB tail (G95–F102) features three Asp residues and a single Glu. In T. vulcanus, no fewer than 8 of the C-terminal 12 residues are negatively charged. Calculations of the electrostatic surface potentials for ThKaiB with and without the C-terminal residues manifest fundamentally different values of the potential (Figure 1E). The importance of the C-terminal region of KaiB was demonstrated by a series of deletion mutants where SeKaiB was truncated one residue at a time starting with SeKaiB:1–100 and ending with SeKaiB:1–94. When these C-terminally truncated proteins were tested in rhythm assays in S. elongatus kaiB-null host cells, the results indicated a strongly weakened and destabilized rhythm for the SeKaiB:1–94 protein (Iwase et al, 2005). Gel-shift-binding assays with full-length SeKaiC, SeKaiB and ThKaiB proteins We used native polyacrylamide gel electrophoresis (PAGE) to assay binding between SeKaiB and ThKaiB with SeKaiC. Both Se proteins were expressed in Escherichia coli using constructs with N-terminal GST tags that were cleaved off prior to PAGE, following previously described procedures with slight modifications (Nishiwaki et al, 2004; Mori et al, 2007). The ThKaiB protein used in the binding assay is identical to the one that served the crystal structure determination and features an N-terminal (His)6-tag. The appearance of bandshift gels did not fundamentally change, irrespective of whether SeKaiC proteins with N- or C-terminal (His)6-tags (data not shown) or the tag-free (GST-off) version were used. Our data demonstrate that both SeKaiB and ThKaiB are able to bind to SeKaiC in the presence of ATP (Figures 2A and B, respectively). Previous observations by our laboratories (Mori et al, 2007) and others (Kageyama et al, 2006) demonstrated that, unlike KaiA, KaiB binds preferably to the P-KaiC hexamer. Binding of ThKaiB to the SeKaiC hexamer is consistent with the observation that ThKaiB partially complements the null KaiB mutant of S. elongatus in vivo, generating a faint rhythm (Iwase et al, 2005). Similarly, KaiB from Anabaena partially complemented the null mutant, whereas KaiB from Synechocystis sp. strain PCC 6803 was able to fully complement it (Iwase et al, 2005). Figure 2.Native PAGE assays for KaiBC complex formation. Binding between (A) full-length SeKaiB and SeKaiC proteins and (B) ThKaiB and SeKaiC proteins. A ‘+’ indicates presence of and a ‘−’ indicates absence of protein (KaiB dimer or SeKaiC hexamer ([SeKaiC]6)). The bands corresponding to KaiBC complexes are indicated with asterisks. Note the considerably different shifts of the SeKaiB and ThKaiB proteins, which are likely due to deviating net charges. ThKaiB has a higher number of acidic residues in the C-terminal tail. The protein amounts used were 30 pmol (ThKaiB and SeKaiC) and 22 pmol (SeKaiB). The buffer for all proteins was 20 mM Tris (pH 7.8), 150 mM NaCl and 1 mM DTT. Only SeKaiC and ThKaiC were supplemented with 5 mM MgCl2 and 1 mM ATP. Download figure Download PowerPoint Binding of KaiB to KaiC fragments The crystal structure of SeKaiC revealed two serially arranged domains (N-terminal KaiCI and C-terminal KaiCII), connected by a 15-amino-acid linker (Pattanayek et al, 2004). Upon oligomerization, these give rise to a double-doughnut-shaped hexamer (featuring KaiCI and KaiCII halves) with a constricted waist in the region of the linkers. KaiA was found to bind to flexible C-terminal peptides (Vakonakis and LiWang, 2004) and may bind more transiently to the KaiCII dome with its six ATP-binding clefts (Pattanayek et al, 2006). However, it is currently not known whether KaiB binds to the KaiCI or the KaiCII half. To examine whether KaiB binds to KaiCI or KaiCII, we expressed the two KaiC domains from S. elongatus separately as GST fusion proteins (CI, residues 1–250, and CII, residues 253–519). The goal was to assay potential formation of a hexameric ring from each of the two fragments to simulate the two halves of the KaiC hexamer. Following affinity chromatography and cleavage of the GST portion, we used native PAGE and negative-stain EM to assay hexamerization of SeKaiCI and SeKaiCII, and tested KaiB binding to either individually expressed KaiC domain. KaiCI in the presence of ATP forms rings by negative-stain EM (Figure 3A). These rings are of the same diameter (100 Å) as observed previously for the hexamer of full-length KaiC (Pattanayek et al, 2006). Native PAGE indicates that KaiCI fails to bind SeKaiB (Figure 3B) or ThKaiB (Supplementary Figure S2A). Figure 3.Oligomerization of KaiC CI and CII fragments and KaiB-binding assays. (A) A negative-stain electron micrograph of the SeKaiCI fragment (aa 1–250) in the presence of ATP. Note the presence of rings ∼100 Å in diameter. The scale bar represents 100 Å. (B) Native PAGE showing that the SeKaiCI fragment oligomerizes in the presence of ATP, but does not bind to SeKaiB. A ‘+’ indicates the presence of and a ‘−’ indicates the absence of protein; the protein amounts used were 30 pmol (SeKaiC and SeKaiCI) and 24 pmol (SeKaiB). (C) A negative-stain electron micrograph of the SeKaiCII fragment (aa 253–519) in the presence of ATP. Note the absence of hexameric rings; the scale bar represents 100 Å. (D) Native PAGE showing that the SeKaiCII binds to SeKaiB. A ‘+’ indicates the presence of and a ‘−’ indicates the absence of protein; 24 pmol SeKaiB and 15 pmol SeKaiCII were used. The band corresponding to a complex between SeKaiB and the SeKaiCII fragment is indicated with an asterisk. (E) SDS–PAGE assay of phosphorylation status of KaiC proteins and SeKaiCI and SeKaiCII. The double bands for ThKaiC, SeKaiC and SeKaiCII indicate that these proteins have both a phosphorylated and an unphosphorylated states. In contrast, the single band for SeKaiCI (lane 5) indicates that it is not phosphorylated. (F) Fluorescence spectra for mixtures of SeKaiA or SeKaiB and a 25mer peptide corresponding to the C-terminal end of SeKaiCII (P494–S519) and labelled with Trp at its C terminus. The concentration of the peptide in the assays was 85 μM. Download figure Download PowerPoint In contrast, the KaiCII fragment did not hexamerize under the same conditions (Figure 3C). This replicates in a second cyanobacterial species the earlier finding by Ishiura et al. who studied hexamer formation with the CI and CII halves from ThKaiC and noted the absence of hexamers for CII. Instead, gel filtration chromatography indicated the presence of either tetramers or pentamers (Hayashi et al, 2006). EM indicates that CII forms irregular oligomers that are smaller than hexamers (Figure 3C). Despite the irregular CII oligomer size, the native PAGE assay reveals that KaiB binds to KaiCII (Figure 3D, SeKaiB, and Supplementary Figure S2B, ThKaiB). It is noteworthy that unlike KaiCI, KaiCII is phosphorylated (Figure 3E) and the protein can be dephosphorylated with λ-phosphatase (Supplementary Figure S3). These data provide evidence that similarly to KaiA, KaiB interacts with the C-terminal ring of the KaiC homo-hexamer. The C-terminal domains of the KaiA dimer bind individual KaiC C-terminal peptides (Vakonakis and LiWang, 2004) and enhance phosphorylation of the hexamer through rapid and repeated action on subunits (Kageyama et al, 2006). To examine whether KaiB also binds the C-terminal KaiC peptide, we tagged a 25mer C-terminal SeKaiC peptide with Trp and measured fluorescence for mixtures of SeKaiB (which lacks Trp) and the KaiC peptide at various ratios (Figure 3F). This fluorescence assay indicates that SeKaiB does not exhibit any affinity for the isolated SeKaiC C-terminal region (residues 495–519). In contrast, the fluorescence assay with SeKaiA and the Trp-labelled SeKaiC peptide confirms KaiA binding to the KaiC C-terminal region (Figure 3F). Negative-stain and cryo-EM analysis of the SeKaiBC complex We have previously used EM to analyse the interactions among the KaiABC proteins during the in vitro circadian cycle (Mori et al, 2007). To help interpret EM images of the observed complexes, we collected 4130 particle images of the negatively stained SeKaiBC complex (Mori et al, 2007). To this we added 4590 particles images of SeKaiBC selected by meta-class sorting from SeKaiABC cycling reactions (Mori et al, 2007), for a total dataset of 8720 particle images. Here, we present a refined 3D structure at ∼19-Å resolution based on these data (Figure 4A). The structure reveals a double-ringed hexameric barrel for KaiC with an additional third layer of density at one end. Assignment of CII as the KaiB contact domain is consistent with our KaiB native PAGE-binding assays (Figure 3 and Supplementary Figure S2). As the reaction mixtures were not purified to defined stoichiometric complexes, the KaiBC structure could represent the average of a heterogeneous population with zero, one, two or three KaiB dimers bound per KaiC hexamer. Therefore, we used the Multirefine program in EMAN to sort the dataset based on five starting model structures (Supplementary Figure S4). These five structures included Ka
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