Quinone sensing by the circadian input kinase of the cyanobacterial circadian clock.

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.

Structure of the N-terminal domain of the circadian clock-associated histidine kinase SasA.

Circadian oscillators are endogenous biological systems that generate the approximately 24 hour temporal pattern of biological processes and confer a reproductive fitness advantage to their hosts. The cyanobacterial clock is the simplest known and the only clock system for which structural information for core component proteins, in this case KaiA, KaiB and KaiC, is available. SasA, a clock-associated histidine kinase, is necessary for robustness of the circadian rhythm of gene expression and implicated in clock output. The N-terminal domain of SasA (N-SasA) interacts directly with KaiC and likely functions as the sensory domain controlling the SasA histidine kinase activity. N-SasA and KaiB share significant sequence similarity and, thus, it has been proposed that they would be structurally similar and may even compete for KaiC binding. Here, we report the NMR structure of N-SasA and show it to be different from that of KaiB. The structural comparisons provide no clear details to suggest competition of SasA and KaiB for KaiC binding. N-SasA adopts a canonical thioredoxin fold but lacks the catalytic cysteine residues. A patch of conserved, solvent-exposed residues is found near the canonical thioredoxin active site. We suggest that this surface is used by N-SasA for protein-protein interactions. Our analysis suggests that the structural differences between N-SasA and KaiB are the result of only a few critical amino acid substitutions.

Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: implications for KaiC regulation.

Circadian clocks are widespread endogenous mechanisms that control the temporal pattern of diverse biological processes, including gene transcription. KaiA is the positive element of the cyanobacterial clock because KaiA overexpression elevates transcription levels of clock components. Recently, we showed that the structure of KaiA is that of a domain-swapped homodimer. The N-terminal domain is a pseudo-receiver; thus, it is likely to be involved in signal transduction in the clock-resetting pathway. The C-terminal domain of KaiA is structurally novel and enhances the KaiC autokinase activity directly. Here, we report the NMR structure of the C-terminal domain of KaiA (ThKaiA180C) in complex with a KaiC-derived peptide from the cyanobacterium Thermosynechococcus elongatus BP-1. The protein-peptide interface is revealed to be different from a model that was proposed earlier, is stabilized by a combination of hydrophobic and electrostatic interactions, and includes many residues known to produce a circadian-period phenotype upon substitution. Although the structure of the monomeric subunit of ThKaiA180C is largely unchanged upon peptide binding, the intersubunit dimerization angle changes. It is proposed that modulation of the C-terminal KaiA domain dimerization angle regulates KaiA-KaiC interactions.

N1...N3 hydrogen bonds of A:U base pairs of RNA are stronger than those of A:T base pairs of DNA.

Trans-hydrogen-bond deuterium isotope effects of Watson-Crick A:U and A:T base pairs of 10 homologous RNA and DNA duplexes are compared. The isotope effect at 13C2 of adenosine residues due to deuterium/protium substitution at the imino H3 site, 2hDelta13C2, is larger in RNA than in DNA. The virtually consistent larger isotope effects in RNA suggest that the N1...N3 hydrogen bonds of A:U base pairs of RNA are stronger than those of the A:T base pairs of DNA.

Crystal structure of circadian clock protein KaiA from Synechococcus elongatus.

The circadian clock found in Synechococcus elongatus, the most ancient circadian clock, is regulated by the interaction of three proteins, KaiA, KaiB, and KaiC. While the precise function of these proteins remains unclear, KaiA has been shown to be a positive regulator of the expression of KaiB and KaiC. The 2.0-A structure of KaiA of S. elongatus reported here shows that the protein is composed of two independently folded domains connected by a linker. The NH(2)-terminal pseudo-receiver domain has a similar fold with that of bacterial response regulators, whereas the COOH-terminal four-helix bundle domain is novel and forms the interface of the 2-fold-related homodimer. The COOH-terminal four-helix bundle domain has been shown to contain the KaiC binding site. The structure suggests that the KaiB binding site is covered in the dimer interface of the KaiA "closed" conformation, observed in the crystal structure, which suggests an allosteric regulation mechanism.

Trans-hydrogen bond deuterium isotope effects of A:T base pairs in DNA.

The chemical shifts of (13)C2 of adenosine residues of DNA were observed to experience a through-space or trans-hydrogen bond isotope effect as a result of deuterium substitution at the imino hydrogen site of base-paired thymidine residues. NMR measurements of several self-complementary DNA duplexes at natural abundance (13)C in 50% H(2)O, 50% D(2)O solvent mixtures yielded an average trans-hydrogen bond isotope effect, (2h)Delta(13)C2, of -47 ppb. The data suggest that stronger hydrogen bonds have more negative (2h)Delta(13)C2 values, which means that A:T N1.H3 hydrogen bonds increase the anharmonicity of the effective vibrational potential of H3. However, (2h)Delta(13)C2 values do not correlate with intra-residue (2)Delta(13)C4 values of thymidine observed here and earlier (Vakonakis et al., 2003), which suggests that (2h)Delta(13)C2 is not determined entirely by hydrogen bond strength. Instead, the variations observed in (2h)Delta(13)C2 values suggest that they may also be sensitive to base pair geometry.

NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: implications for KaiA-KaiC interaction.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all alpha-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-A-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment.

Internal motions of d-ribose selectively 2H-labeled at the 2' position were measured using solid state 2H NMR experiments. A sample of d-ribose-2'-d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of -74 and -60 degrees C the C2'-H2' bond was observed to undergo two kinds of motions which were similar to those of C2'-H2'/H2" found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609-8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6+/-0.7 kcal mol(-1), and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy > or =4 kcal mol(-1). At -74 degrees C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at -60 degrees C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2'-H2' bond are most likely from interconversions between C2'-endo and C3'-endo by way of the O4'-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2'-H2' bond within the C2'-endo and C3'-endo potential energy minima.

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA.

Deuterium isotope effects and fractionation factors of N1.H3-N3 hydrogen bonded Watson-Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of (13)C2 and (13)C4, (2)delta(13)C2 and (2)delta(13)C4, and equilibrium deuterium/protium fractionation factors of H3, Phi, were measured and seen to correlate with the chemical shift of the corresponding imino proton, delta(H3). Downfield-shifted imino protons associated with larger values of (2)delta(13)C2 and (2)delta(13)C4 and smaller Phi values, which together suggested that the effective H3-N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that (2)delta(13)C2, (2)delta(13)C4 and Phi values can be useful gauges of hydrogen bond strength of A:T base pairs.

Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.

In the cyanobacterium Synechococcus elongatus (PCC 7942) the proteins KaiA, KaiB, and KaiC are required for circadian clock function. We deduced a circadian clock function for KaiA from a combination of biochemical and structural data. Both KaiA and its isolated carboxyl-terminal domain (KaiA180C) stimulated KaiC autophosphorylation and facilitated attenuation of KaiC autophosphorylation by KaiB. An amino-terminal domain (KaiA135N) had no function in the autophosphorylation assay. NMR structure determination showed that KaiA135N is a pseudo-receiver domain. We propose that this pseudo-receiver is a timing input-device that regulates KaiA stimulation of KaiC autophosphorylation, which in turn is essential for circadian timekeeping.