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Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.
Johnson CH, Egli M
(2014) Annu Rev Biochem 83: 221-47
MeSH Terms: Bacterial Proteins, Circadian Clocks, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Cyanobacteria, Feedback, Physiological, Gene Expression Regulation, Bacterial, Homeostasis, Protein Biosynthesis, Protein Processing, Post-Translational, Temperature, Transcription, Genetic
Show Abstract · Added June 26, 2014
For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.
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1 Members
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12 MeSH Terms
An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
Pattanayek R, Xu Y, Lamichhane A, Johnson CH, Egli M
(2014) Acta Crystallogr D Biol Crystallogr 70: 1375-90
MeSH Terms: Adenosine Triphosphatases, Arginine, Bacterial Proteins, Binding Sites, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Models, Molecular, Mutagenesis, Site-Directed, Phosphorylation, Protein Structure, Tertiary, Synechococcus
Show Abstract · Added May 27, 2014
A post-translational oscillator (PTO) composed of the proteins KaiA, KaiB and KaiC is at the heart of the cyanobacterial circadian clock. KaiC interacts with KaiA and KaiB over the daily cycle, and CII domains undergo rhythmic phosphorylation/dephosphorylation with a 24 h period. Both the N-terminal (CI) and C-terminal (CII) rings of KaiC exhibit ATPase activity. The CI ATPase proceeds in an input-independent fashion, but the CII ATPase is subject to metabolic input signals. The crystal structure of KaiC from Thermosynechococcus elongatus allows insight into the different anatomies of the CI and CII ATPases. Four consecutive arginines in CI (Arg linker) that connect the P-loop, CI subunits and CI and CII at the ring interface are primary candidates for the coordination of the CI and CII activities. The mutation of linker residues alters the period or triggers arhythmic behavior. Comparison between the CI and CII structures also reveals differences in loop regions that are key to KaiA and KaiB binding and activation of CII ATPase and kinase. Common packing features in KaiC crystals shed light on the KaiB-KaiC interaction.
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2 Members
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11 MeSH Terms
Circadian yin-yang regulation and its manipulation to globally reprogram gene expression.
Xu Y, Weyman PD, Umetani M, Xiong J, Qin X, Xu Q, Iwasaki H, Johnson CH
(2013) Curr Biol 23: 2365-74
MeSH Terms: Bacterial Proteins, CLOCK Proteins, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Gene Expression, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Hydrogen, Hydrogenase, Multigene Family, Phosphorylation, Promoter Regions, Genetic, Synechococcus, Transcription, Genetic
Show Abstract · Added February 12, 2015
BACKGROUND - The cyanobacterial circadian program exerts genome-wide control of gene expression. KaiC undergoes rhythms of phosphorylation that are regulated by interactions with KaiA and KaiB. The phosphorylation status of KaiC is thought to mediate global transcription via output factors SasA, CikA, LabA, RpaA, and RpaB. Overexpression of kaiC has been reported to globally repress gene expression.
RESULTS - Here, we show that the positive circadian component KaiA upregulates "subjective dusk" genes and that its overexpression deactivates rhythmic gene expression without significantly affecting growth rates in constant light. We analyze the global patterns of expression that are regulated by KaiA versus KaiC and find in contrast to the previous report of KaiC repression that there is a "yin-yang" regulation of gene expression whereby kaiA overexpression activates "dusk genes" and represses "dawn genes," whereas kaiC overexpression complementarily activates dawn genes and represses dusk genes. Moreover, continuous induction of kaiA latched KaiABC-regulated gene expression to provide constitutively increased transcript levels of diverse endogenous and heterologous genes that are expressed in the predominant subjective dusk phase. In addition to analyzing KaiA regulation of endogenous gene expression, we apply these insights to the expression of heterologous proteins whose products are of potential value, namely human proinsulin, foreign luciferase, and exogenous hydrogenase.
CONCLUSIONS - Both KaiC and KaiA complementarily contribute to the regulation of circadian gene expression via yin-yang switching. Circadian patterns can be reprogrammed by overexpression of kaiA or kaiC to constitutively enhance gene expression, and this reprogramming can improve 24/7 production of heterologous proteins that are useful as pharmaceuticals or biofuels.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates that KaiB monomers interact with KaiC and block ATP binding clefts.
Villarreal SA, Pattanayek R, Williams DR, Mori T, Qin X, Johnson CH, Egli M, Stewart PL
(2013) J Mol Biol 425: 3311-24
MeSH Terms: Adenosine Triphosphate, Bacterial Proteins, Binding Sites, Circadian Rhythm Signaling Peptides and Proteins, Cryoelectron Microscopy, Crystallography, X-Ray, Models, Molecular, Molecular Dynamics Simulation, Multiprotein Complexes, Phosphorylation, Protein Binding, Protein Multimerization, Protein Structure, Quaternary, Synechococcus
Show Abstract · Added March 7, 2014
The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB-KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
Non-optimal codon usage is a mechanism to achieve circadian clock conditionality.
Xu Y, Ma P, Shah P, Rokas A, Liu Y, Johnson CH
(2013) Nature 495: 116-20
MeSH Terms: Bacterial Proteins, Circadian Clocks, Circadian Rhythm, Circadian Rhythm Signaling Peptides and Proteins, Codon, Gene Expression Regulation, Bacterial, Genes, Bacterial, Multigene Family, Phenotype, Selection, Genetic, Synechococcus, Temperature
Show Abstract · Added May 30, 2014
Circadian rhythms are oscillations in biological processes that function as a key adaptation to the daily rhythms of most environments. In the model cyanobacterial circadian clock system, the core oscillator proteins are encoded by the gene cluster kaiABC. Genes with high expression and functional importance, such as the kai genes, are usually encoded by optimal codons, yet the codon-usage bias of the kaiBC genes is not optimized for translational efficiency. We discovered a relationship between codon usage and a general property of circadian rhythms called conditionality; namely, that endogenous rhythmicity is robustly expressed under some environmental conditions but not others. Despite the generality of circadian conditionality, however, its molecular basis is unknown for any system. Here we show that in the cyanobacterium Synechococcus elongate, non-optimal codon usage was selected as a post-transcriptional mechanism to switch between circadian and non-circadian regulation of gene expression as an adaptive response to environmental conditions. When the kaiBC sequence was experimentally optimized to enhance expression of the KaiB and KaiC proteins, intrinsic rhythmicity was enhanced at cool temperatures that are experienced by this organism in its natural habitat. However, fitness at those temperatures was highest in cells in which the endogenous rhythms were suppressed at cool temperatures as compared with cells exhibiting high-amplitude rhythmicity. These results indicate natural selection against circadian systems in cyanobacteria that are intrinsically robust at cool temperatures. Modulation of circadian amplitude is therefore crucial to its adaptive significance. Moreover, these results show the direct effects of codon usage on a complex phenotype and organismal fitness. Our work also challenges the long-standing view of directional selection towards optimal codons, and provides a key example of natural selection against optimal codons to achieve adaptive responses to environmental changes.
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12 MeSH Terms
Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
Pattanayek R, Yadagiri KK, Ohi MD, Egli M
(2013) Cell Cycle 12: 810-7
MeSH Terms: Bacterial Proteins, Circadian Clocks, Circadian Rhythm Signaling Peptides and Proteins, Histidine, Models, Molecular, Nanostructures, Negative Staining, Oligopeptides, Protein Binding, Scattering, Small Angle, Static Electricity, Synechococcus, X-Ray Diffraction
Show Abstract · Added March 7, 2014
In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.
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2 Members
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13 MeSH Terms
Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
Egli M, Pattanayek R, Sheehan JH, Xu Y, Mori T, Smith JA, Johnson CH
(2013) Biochemistry 52: 1208-20
MeSH Terms: Bacterial Proteins, Circadian Clocks, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Models, Molecular, Molecular Dynamics Simulation, Mutation, Phosphorylation, Protein Conformation, Protein Multimerization, Synechococcus, Thermodynamics, Valine
Show Abstract · Added May 30, 2013
The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.
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3 Members
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13 MeSH Terms
Crystal structure of the redox-active cofactor dibromothymoquinone bound to circadian clock protein KaiA and structural basis for dibromothymoquinone's ability to prevent stimulation of KaiC phosphorylation by KaiA.
Pattanayek R, Sidiqi SK, Egli M
(2012) Biochemistry 51: 8050-2
MeSH Terms: Amino Acid Sequence, Bacterial Proteins, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Dibromothymoquinone, Dimerization, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Oxidation-Reduction, Phosphorylation, Protein Conformation
Show Abstract · Added March 7, 2014
KaiA protein that stimulates KaiC phosphorylation in the cyanobacterial circadian clock was recently shown to be destabilized by dibromothymoquinone (DBMIB), thus revealing KaiA as a sensor of the plastoquinone (PQ) redox state and suggesting an indirect control of the clock by light through PQ redox changes. Here we show using X-ray crystallography that several DBMIBs are bound to KaiA dimer. Some binding modes are consistent with oligomerization of N-terminal KaiA pseudoreceiver domains and/or reduced interdomain flexibility. DBMIB bound to the C-terminal KaiA (C-KaiA) domain and limited stimulation of KaiC kinase activity by C-KaiA in the presence of DBMIB demonstrate that the cofactor may weakly inhibit KaiA-KaiC binding.
0 Communities
1 Members
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11 MeSH Terms
Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH
(2012) Biochemistry 51: 1547-58
MeSH Terms: Adenosine Triphosphatases, Amino Acid Sequence, Bacterial Proteins, CLOCK Proteins, Catalytic Domain, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Cyanobacteria, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation
Show Abstract · Added March 7, 2014
The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.
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2 Members
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12 MeSH Terms
Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
Pattanayek R, Williams DR, Rossi G, Weigand S, Mori T, Johnson CH, Stewart PL, Egli M
(2011) PLoS One 6: e23697
MeSH Terms: Bacterial Proteins, Binding Sites, Circadian Clocks, Circadian Rhythm Signaling Peptides and Proteins, Crystallography, X-Ray, Histidine Kinase, Microscopy, Electron, Phosphotransferases, Protein Conformation, Protein Interaction Domains and Motifs, Protein Kinases, Scattering, Small Angle, Synechococcus, X-Ray Diffraction
Show Abstract · Added March 7, 2014
The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.
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14 MeSH Terms