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.

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.

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.

Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding.

Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.

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.

Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis.

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase.

In the mammalian endoplasmic reticulum, oxidoreductin-1α (Ero1α) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O2 to H2O2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H2O2 generation cause cell death, Ero1α activity must be properly regulated. In addition to the four catalytic cysteines (Cys94, Cys99, Cys104, and Cys131) that are located in the flexible active site region, the Cys208-Cys241 pair located at the base of another flexible loop is necessary for Ero1α regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys208-Cys241 disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys208 and Cys241 did not grossly affect the thermal stability or overall shape of Ero1α, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys208 and Cys241 provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1α through reduction of the Cys208-Cys241 disulfide. We propose a mechanism of dual Ero1α regulation by dynamic redox interactions between PDI and the two Ero1α flexible loops that harbor the regulatory cysteines.

Assembly and disassembly dynamics of the cyanobacterial periodosome.

In vitro incubation of three Kai proteins, KaiA, KaiB, and KaiC, with ATP induces a KaiC phosphorylation cycle that is a potential circadian clock pacemaker in cyanobacterium Synechococcus elongatus PCC 7942. The Kai proteins assemble into large heteromultimeric complexes (periodosome) to effect a robust oscillation of KaiC phosphorylation. Here, we report real-time measurements of the assembly/disassembly dynamics of the Kai periodosome by using small-angle X-ray scattering and determination of the low-resolution shapes of the KaiA:KaiC and KaiB:KaiC complexes. Most previously identified period-affecting mutations could be mapped to the association interfaces of our complex models. Our results suggest that the assembly/disassembly processes are crucial for phase entrainment in the early synchronizing stage but are passively driven by the phosphorylation status of KaiC in the late oscillatory stage. The Kai periodosome is assembled in such a way that KaiA and KaiB are recruited to a C-terminal region of KaiC in a phosphorylation-dependent manner.

Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution.

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring. During the phosphorylation cycle, the C-terminal domains of KaiC are repositioned in a stepwise manner to affect global expansion and contraction motions of the C-terminal ring. Arg393 of KaiC has a critical function in expanding the C-terminal ring and its replacement with Cys affects the temperature compensation of the period--a fundamental property of circadian clocks. The conformational ticking of KaiC observed here in solution serves as a timing cue for assembly/disassembly of other clock proteins (KaiA and KaiB), and is interlocked with its auto-inhibitory ATPase underlying circadian periodicity of cyanobacteria.

Structure-function relationship of KaiC around dawn.

KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.

Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.

Structural and dynamic aspects of protein clocks: how can they be so slow and stable?

KaiC is a core protein of the cyanobacterial Kai oscillator, which persists without transcription-translation feedback. In the presence of KaiA and KaiB, KaiC reveals rhythmic activation/inactivation of its ATPase and autokinase/autophosphotase activities over approximately 24 h. Since the in vitro reconstruction of the Kai oscillator, the structures and functions of the Kai proteins have been studied extensively. Each protein's crystal structure and low-resolution views of Kai complexes have been reported. In addition, newer data are emerging on dynamic aspects such as assembly/disassembly of the Kai components and a ticking motion of KaiC, which is probably coupled to its slow, temperature-compensated ATPase activity. The accumulated evidence offers an ideal opportunity to revisit a fundamental question regarding biological circadian clocks: what determines the temperature-compensated 24 h period? In this review, I summarize the current understanding of the Kai oscillator's molecular mechanism and discuss emerging ideas on protein clocks.

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.

Adenylate kinase 1 overexpression increases locomotor activity in medaka fish.

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

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.

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR.

The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization.

P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.

Biophysical research in Okazaki, Japan.

This commentary summarizes the recent biophysical research conducted at the National Institute for Basic Biology, the National Institute for Physiological Sciences, and the Institute for Molecular Science in Okazaki, Japan.

Oxidative misfolding of Cu/Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation.

Misfolded Cu/Zn-superoxide dismutase (SOD1) is a pathological species in a subset of amyotrophic lateral sclerosis (ALS). Oxidative stress is known to increase in affected spinal cords of ALS and is thus considered to cause damages on SOD1 leading to the misfolding and aggregation. Despite this, it still remains elusive what triggers misfolding of SOD1 under oxidizing environment. Here, we show that a thiol group of Cys111 in SOD1 is oxidized to a sulfenic acid with hydrogen peroxide and reveal that further dissociation of the bound metal ions from the oxidized SOD1 allows another free Cys residue (Cys6) to nucleophilically attack the sulfenylated Cys111. As a result, an intra-molecular disulfide bond forms between Cys6 and Cys111. Such an abnormal SOD1 with the non-canonical disulfide bond was conformationally extended with significant cytotoxicity as well as high propensity to aggregate. Taken together, we propose a new model of SOD1 misfolding under oxidizing environment, in which formation of the non-canonical intramolecular disulfide bond plays a pivotal role.

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.