Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix-coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

Slow and temperature-compensated autonomous disassembly of KaiB-KaiC complex.

KaiC is the central pacemaker of the circadian clock system in cyanobacteria and forms the core in the hetero-multimeric complexes, such as KaiB-KaiC and KaiA-KaiB-KaiC. Although the formation process and structure of the binary and ternary complexes have been studied extensively, their disassembly dynamics have remained elusive. In this study, we constructed an experimental system to directly measure the autonomous disassembly of the KaiB-KaiC complex under the condition where the dissociated KaiB cannot reassociate with KaiC. At 30°C, the dephosphorylated KaiB-KaiC complex disassembled with an apparent rate of 2.1±0.3 d-1, which was approximately twice the circadian frequency. Our present analysis using a series of KaiC mutants revealed that the apparent disassembly rate correlates with the frequency of the KaiC phosphorylation cycle in the presence of KaiA and KaiB and is robustly temperature-compensated with a Q 10 value of 1.05±0.20. The autonomous cancellation of the interactions stabilizing the KaiB-KaiC interface is one of the important phenomena that provide a link between the molecular-scale and system-scale properties.

Regulation mechanisms of the dual ATPase in KaiC.

KaiC is a dual adenosine triphosphatase (ATPase), with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual-ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to adenosine triphosphate (ATP) is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The up-regulatory effect on the N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB.

Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5'-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.

Tuning the circadian period of cyanobacteria up to 6.6 days by the single amino acid substitutions in KaiC.

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.

Author Correction: Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria.

The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.

KaiC from a cyanobacterium Gloeocapsa sp. PCC 7428 retains functional and structural properties required as the core of circadian clock system.

KaiC, the core protein of the cyanobacterial clock, assembles into a hexamer upon ATP-binding. The hexameric KaiC from a cyanobacterium Synechococcus elongatus PCC 7942 (Se-KaiC) is a multifunctional enzyme with autokinase, autophosphatase and ATPase and these activities show a circadian rhythm in the presence of two other clock proteins, KaiA and KaiB both in vivo and in vitro. While an interplay among three enzymatic activities has been pointed out through studies on Se-KaiC as the basis of circadian rhythmicity in cyanobacteria, little is known about the structure and functions of KaiC from other cyanobacterial species. In this study, we established a protocol to prepare KaiC from Gloeocapsa sp. PCC 7428 (Gl-KaiC) belonging to a distinct genus from Synechococcus and characterized its oligomeric structure and function. The results demonstrate that Gl-KaiC shares the basic properties with Se-KaiC. The present protocol offers practical means for further analysis of structure and function of Gl-KaiC, which would provide insights into diversity and evolution of the clock systems in cyanobacteria.

Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyclic phosphorylation and dephosphorylation at Ser431 and Thr432 in the C2 domain proceed with a period of approximately 24 h in the presence of other clock proteins, KaiA and KaiB, but recent studies have revealed a crucial role for the C1 ring in determining the cycle period. In this study, we mapped dynamic structural changes of the C1 ring in solution using a combination of site-directed tryptophan mutagenesis and fluorescence spectroscopy. We found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure. This transition triggered by ATP hydrolysis in the C1 ring in specific phosphorylation states is a necessary event for recruitment of KaiB, limiting the overall rate of slow complex formation. Our results provide structural and kinetic insights into the C1-ring rearrangements governing the slow dynamics of the cyanobacterial circadian clock.

Accelerating in vitro studies on circadian clock systems using an automated sampling device.

KaiC, a core protein of the cyanobacterial circadian clock, is rhythmically autophosphorylated and autodephosphorylated with a period of approximately 24 h in the presence of two other Kai proteins, KaiA and KaiB. In vitro experiments to investigate the KaiC phosphorylation cycle consume considerable time and effort. To automate the fractionation, quantification, and evaluation steps, we developed a suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software. Eight sample tables can be controlled independently at different temperatures within a fluctuation of ±0.01°C, enabling investigation of the temperature dependency of clock activities simultaneously in a single experiment. The suite includes an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability. Multiple lanes on a gel can be detected quasi-automatically through an auto-detection procedure implemented in the software, with or without correction for lane 'smiling.' To demonstrate the performance of the suite, robustness of the period against temperature variations was evaluated using 32 datasets of the KaiC phosphorylation cycle. By using the software, the time required for the analysis was reduced by approximately 65% relative to the conventional method, with reasonable reproducibility and quality. The suite is potentially applicable to other clock or clock-related systems in higher organisms, relieving users from having to repeat multiple manual sampling and analytical steps.

ADP-Ribose Pyrophosphatase Reaction in Crystalline State Conducted by Consecutive Binding of Two Manganese(II) Ions as Cofactors.

Adenosine diphosphate ribose pyrophosphatase (ADPRase), a member of the Nudix family proteins, catalyzes the metal-induced and concerted general acid-base hydrolysis of ADP ribose (ADPR) into AMP and ribose-5'-phosphate (R5P). The ADPR-hydrolysis reaction of ADPRase from Thermus thermophilus HB8 (TtADPRase) requires divalent metal cations such as Mn(2+), Zn(2+), or Mg(2+) as cofactors. Here, we report the reaction pathway observed in the catalytic center of TtADPRase, based on cryo-trapping X-ray crystallography at atomic resolutions around 1.0 Å using Mn(2+) as the reaction trigger, which was soaked into TtADPRase-ADPR binary complex crystals. Integrating 11 structures along the reaction timeline, five reaction states of TtADPRase were assigned, which were ADPRase alone (E), the ADPRase-ADPR binary complex (ES), two ADPRase-ADPR-Mn(2+) reaction intermediates (ESM, ESMM), and the postreaction state (E'). Two Mn(2+) ions were inserted consecutively into the catalytic center of the ES-state and ligated by Glu86 and Glu82, which are highly conserved among the Nudix family, in the ESM- and ESMM-states. The ADPR-hydrolysis reaction was characterized by electrostatic, proximity, and orientation effects, and by preferential binding for the transition state. A new reaction mechanism is proposed, which differs from previous ones suggested from structure analyses with nonhydrolyzable substrate analogues or point-mutated ADPRases.