Yong-Gang Chang

A protein fold switch joins the circadian oscillator to clock output in cyanobacteria

Biochemical basis of a 24-hour clock Circadian clocks keep organisms in synch with such daily cycles as illumination, activity, and food availability. The circadian clock in cyanobacteria has the necessary 24-hour period despite its three component proteins having biochemical activities that occur on a much faster time scale. Abe et al. focused on the cyanobacterial clock component KaiC, an adenosine triphosphatase (ATPase) that can autophosphorylate and autodephosphorylate. The slow ATPase activity of KaiC, which is linked to a peptide isomerisation, provided the slow kinetics that set the speed of the 24-hour clock. Chang et al. found that another clock component, KaiB, also has slow changes in its protein conformation that help to set the oscillation period of the clock and its signaling output. Science, this issue pp. 312 and 324 Slow conformational change of a protein helps set the pace of a circadian clock. Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of Earth’s rotation. Once KaiB switches folds, it binds phosphorylated KaiC and captures KaiA, which initiates a phase transition of the circadian cycle, and it regulates components of the clock-output pathway, which provides the link that joins the timekeeping and signaling functions of the oscillator.

Rhythmic ring–ring stacking drives the circadian oscillator clockwise

The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a self-sustained ∼24-h rhythm of phosphorylation of KaiC. The mechanism propelling this oscillator has remained elusive, however. We show that stacking interactions between the CI and CII rings of KaiC drive the transition from the phosphorylation-specific KaiC–KaiA interaction to the dephosphorylation-specific KaiC–KaiB interaction. We have identified the KaiB-binding site, which is on the CI domain. This site is hidden when CI domains are associated as a hexameric ring. However, stacking of the CI and CII rings exposes the KaiB-binding site. Because the clock output protein SasA also binds to CI and competes with KaiB for binding, ring stacking likely regulates clock output. We demonstrate that ADP can expose the KaiB-binding site in the absence of ring stacking, providing an explanation for how it can reset the clock.

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

In the cyanobacterial circadian oscillator, KaiA and KaiB alternately stimulate autophosphorylation and autodephosphorylation of KaiC with a periodicity of approximately 24 h. KaiA activates autophosphorylation by selectively capturing the A loops of KaiC in their exposed positions. The A loops and sites of phosphorylation, residues S431 and T432, are located in the CII ring of KaiC. We find that the flexibility of the CII ring governs the rhythm of KaiC autophosphorylation and autodephosphorylation and is an example of dynamics-driven protein allostery. KaiA-induced autophosphorylation requires flexibility of the CII ring. In contrast, rigidity is required for KaiC-KaiB binding, which induces a conformational change in KaiB that enables it to sequester KaiA by binding to KaiA’s linker. Autophosphorylation of the S431 residues around the CII ring stabilizes the CII ring, making it rigid. In contrast, autophosphorylation of the T432 residues offsets phospho-S431-induced rigidity to some extent. In the presence of KaiA and KaiB, the dynamic states of the CII ring of KaiC executes the following circadian rhythm: . Apparently, these dynamic states govern the pattern of phosphorylation, ST → SpT → pSpT → pST → ST. CII-CI ring-on-ring stacking is observed when the CII ring is rigid, suggesting a mechanism through which the ATPase activity of the CI ring is rhythmically controlled. SasA, a circadian clock-output protein, binds to the CI ring. Thus, rhythmic ring stacking may also control clock-output pathways.

Structural basis of the day-night transition in a bacterial circadian clock

Molecular clockwork from cyanobacteria The cyanobacterial circadian clock oscillator can be reconstituted in a test tube from just three proteins—KaiA, KaiB, and KaiC—and adenosine triphosphate (ATP). Tseng et al. studied crystal and nuclear magnetic resonance structures of complexes of the oscillator proteins and their signaling output proteins and tested the in vivo effects of structure-based mutants. Large conformational changes in KaiB and ATP hydrolysis by KaiC are coordinated with binding to output protein, which couples signaling and the day-night transitions of the clock. Snijder et al. provide complementary analysis of the oscillator proteins by mass spectrometry and cryo–electron microscopy. Their results help to explain the structural basis for the dynamic assembly of the oscillator complexes. Science, this issue p. 1174, p. 1181 Cyanobacteria make a clock from just three proteins. Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

Cooperative KaiA–KaiB–KaiC Interactions Affect KaiB/SasA Competition in the Circadian Clock of Cyanobacteria

The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB-KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB-KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA-KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA-KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiBs favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.

Analysis of the Helium Behavior Due to AC Losses in the KSTAR Superconducting Coils

The KSTAR superconducting magnetic coils, which are made of cable in-conduit conductor (CICC), maintain a superconducting state with forced-flow supercritical helium (4.5 K, 5.5 bar). During current changing of the superconducting magnetic coils, AC losses are generated in the CICC due to dl/dt, and the heat generated from the loss is removed by high heat capacity supercritical helium. At the same time, reversed flow of the helium occurs due to a rapid increase of the helium temperature and momentary changing of the pressure inside the CICC. This phenomenon has been detected in all of the poloidal field (PF) coils, especially in the upper (U) and lower (L) PF1~PF4 coils. The maximum change of the magnetic field in the PF1UL~PF4UL coils is located near the inlet and outlet of the helium cooling channels, and that of the PF5UL~7UL coils is located at the center of the cooling channel. The temperature variation at the helium inlet was always measured to have a time delay after each shot. In the PF1 coil tests, it was measured to have a delay of 26 sec. During the first plasma campaign, this phenomenon was more severe in the case of all PF coils operating together than for a single PF operation. In this paper, we investigated the thermal-hydraulics of this phenomenon.

First Commissioning Results of the KSTAR Cryogenic System

The cryogenic system for the KSTAR superconducting (SC) magnets has been commissioned. It consists of a cold box, distribution boxes (DB) and cryogenic transfer lines. The cold box and DB #1 provide 600 g/s of supercritical helium to cool the SC magnets, their SC bus-lines, and the magnet support structures. It also provides 17.5 g/s of liquid helium to the current leads and supplies cold helium flow to the thermal shields. The main duties of the DB #2 are the relative distribution of the cryogenic helium among the cooling channels of each KSTAR cold component and the emergency release of over-pressurized helium during abnormal events such as quenches of the SC magnets. After individual commissioning, the system was integrated and cooled down with the KSTAR device. In this paper, the construction and commissioning results of the KSTAR cryogenic system will be introduced. In addition, we will present the cool-down results of the KSTAR device.

Construction and Commissioning of the KSTAR Current Feeder System

The function of the current feeder system (CFS) is for conducting large currents from the power supplies to the KSTAR superconducting (SC) magnets. The CFS consists of SC bus-lines, joints, and current leads. The bus-line conductor is a circular cable-in-conduit conductor (CICC), which consists of a 4.5 mm thick stainless steel 316L seamless pipe containing 324 strands of chrome coated NbTi superconductor and 243 strands of OFHC. The ends of the CICC are assembled with specially designed lap joints. The joining resistance is controlled to less than 2.5 nano-ohm to minimize Joule heating. The outer surfaces of the CICC were electrically insulated up to 15 kV with jackets made of Kapton film and prepreged E-glass tape. Helically wrapped conducting fiber was used to measure the voltages of bus-line quenches. Two pairs of prototype brass leads for poloidal field (PF) and toroidal field (TF) coils have been fabricated and tested up to the currents of 26 kA for the PF leads and 35 kA for the TF leads. The test results satisfied all the requirements so that all 18 leads were manufactured and assembled on site. This paper will describe the detailed manufacturing progress and commissioning results of the KSTAR CFS.

Construction and Commissioning of the KSTAR Helium Distribution System

The KSTAR components requiring cryogenic helium coolant for superconducting magnet operations are connected to the helium refrigeration system (HRS) through the helium distribution system (HDS), the final helium distribution station. Twenty eight cryogenic valves including 4 quench protecting valves, many sensors such as temperature sensors, pressure transmitters, and flowmeters are mounted on the system. The HDS has to control the 4.5 K supercritical helium (600 g/s) for 30 superconducting coils and their superconducting bus-lines, 55 K helium (270 g/s) pressurized to 18 bars for the thermal shields and the gravity supports, and a maximum 17.5 g/s of liquid helium for 18 current leads, provided from the HRS which has a 4.5 K equivalent cooling power of 9 kW. The helium distribution system consists of a helium distribution box (DB), 5 helium transfer lines, a PLC based helium control system (HCS), etc. The helium control system is connected to the KSTAR supervisory control & interlock system. The detailed status regarding the construction, commissioning during first cool-down, and the instrumentation & control (I&C) system are included in this paper.

Stability Analysis of the KSTAR PF Busline

The Cable-In-Conduit Conductor (CICC) for the KSTAR buslines is made of NbTi superconducting (SC) strands. A busline consists of several electrical joints, which are the major heat load contributors to the busline cryo-system. In the poloidal field (PF) busline helium circuit, the supercritical helium is fed to the electrical joint of current lead end and comes out to the magnet terminal joint. This helium flow configuration has been verified to maintain the cryogenic stability of the buslines through the KSTAR operation. During the normal operation of the KSTAR PF coil, the heated helium coming out to both the coil and the busline meets at the magnet terminal and exchange heat, but the busline outlet temperature still remained less than magnet outlet temperature. As the buslines for the electrical connection in series of the upper and lower coils for PF1 and PF2 have the helium path through the two terminal joints of magnet, they experience higher temperature than the other buslines mainly due to the larger heat exchange. In this case, the connection buslines are considered to have very low safety margin and have the strong possibility of quenches.