ABSTRACT
The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Circadian Rhythm Signaling Peptides and Proteins/chemistry , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Synechococcus/metabolism , Bacterial Proteins/genetics , Binding Sites , Circadian Rhythm/physiology , Circadian Rhythm Signaling Peptides and Proteins/genetics , Dibromothymoquinone/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Phosphorylation , Protein Kinases/chemistry , Protein Kinases/metabolism , Protein Multimerization , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Synechococcus/geneticsSubject(s)
Acyltransferases/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Ankyrin Repeat , Nerve Tissue Proteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray/methods , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
Huntington and related neurological diseases result from expansion of a polyglutamine (polyQ) tract. The linear lattice model for the structure and binding properties of polyQ proposes that both expanded and normal polyQ tracts in the preaggregation state are random-coil structures but that an expanded polyQ repeat contains a larger number of epitopes recognized by antibodies or other proteins. The crystal structure of polyQ bound to MW1, an antibody against polyQ, reveals that polyQ adopts an extended, coil-like structure. Consistent with the linear lattice model, multimeric MW1 Fvs bind more tightly to longer than to shorter polyQ tracts and, compared with monomeric Fv, bind expanded polyQ repeats with higher apparent affinities. These results suggest a mechanism for the toxicity of expanded polyQ and a strategy to link anti-polyQ compounds to create high-avidity therapeutics.
Subject(s)
Antigen-Antibody Complex/chemistry , Peptides/chemistry , Peptides/immunology , Antibodies/immunology , Antibody Affinity , Crystallography, X-Ray , Immunoglobulin Fragments , Models, Chemical , Neurodegenerative Diseases/etiology , Peptides/toxicity , Protein Binding , Protein Conformation , Protein Structure, SecondaryABSTRACT
The circadian input kinase (CikA) is a major element of the pathway that provides environmental information to the circadian clock of the cyanobacterium Synechococcus elongatus. CikA is a polypeptide of 754 residues and has three recognizable domains: GAF, histidine protein kinase, and receiver-like. This latter domain of CikA lacks the conserved phospho-accepting aspartyl residue of bona fide receiver domains and is thus a pseudo-receiver (PsR). Recently, it was shown that the PsR domain (1) attenuates the autokinase activity of CikA, (2) is necessary to localize CikA to the cell pole, and (3) is necessary for the destabilization of CikA in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The solution structure of the PsR domain of CikA, CikAPsR, is presented here. A model of the interaction between the PsR domain and HPK portion of CikA provides a potential explanation for how the PsR domain attenuates the autokinase activity of CikA. Finally, a likely quinone-binding surface on CikAPsR is shown here.
Subject(s)
Bacterial Proteins/chemistry , Protein Kinases/chemistry , Synechococcus/enzymology , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Structure, Tertiary , SolutionsABSTRACT
Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.
