The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a selfsustained ∼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.dynamic allostery | NMR | protein structure | kinase | kinetics

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: CII ST flexible → CII SpT flexible → CII pSpT rigid → CII pST very-rigid → CII ST flexible . 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.

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 KaiB’s 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.

Bone implants with surface modifications that promote the physiological activities of osteoblasts are the first step for osseointegration in bone repair. Hydroxyapatite is the main inorganic component in mammal bones and teeth, and nanoscaled hydroxyapatite promotes the adhesion of osteoblastic cells. In this study, we created a nano/micro hierarchical structure using micro-arc oxidation coatings and hydrothermal treatments at 150 °C, 175 °C, and 200 °C for 2, 6, 12, and 24 h. After undergoing hydrothermal treatment for 24 h, CaTiO3 began forming regular-shaped crystals at the surface at 175 °C. In order to decrease the CaTiO3 formations and increase the apatite fabrication, a shorter time of hydrothermal treatment was required at 175 °C. There was still surface damage on samples treated for 6 h at 175 °C; however, the nano/micro hierarchical structures were formed in 2 h at 175 °C. The normalized alkaline phosphatase (ALP) activities of the MC3T3-E1 cells with micro-arc oxidation (MAO) coatings and nano/micro hierarchical bioceramics coatings were 4.51 ± 0.26 and 7.36 ± 0.51 μmol p-NP/mg protein (*** P value of <0.001), respectively. The MC3T3-E1 cells with coatings showed highly statistically significant results in terms of the ALP activity. This proposed nano/micro hierarchical structure promoted cell proliferation and osteogenic differentiation of the osteoblast MC3T3-E1 cells. This study realized a promising nano system for osseointegration via bone implant surface treatments, which can promote the physiological activities of osteoblasts.