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.
FliG, which is composed of three distinctive domains, N-terminal (N), middle (M), and C-terminal (C), is an essential rotor component that generates torque and determines rotational direction. To determine the role of FliG in determining flagellar rotational direction, we prepared rotational biased mutants of fliG in Vibrio alginolyticus. The E144D mutant, whose residue is belonging to the EHPQR-motif in FliGM, exhibited an increased number of switching events. This phenotype generated a response similar to the phenol-repellent response in chemotaxis. To clarify the effect of E144D mutation on the rotational switching, we combined the mutation with other che mutations (G214S, G215A and A282T) in FliG. Two of the double mutants suppressed the rotational biased phenotype. To gain structural insight into the mutations, we performed molecular dynamic simulations of the FliGMC domain, based on the crystal structure of Thermotoga maritima FliG and nuclear magnetic resonance analysis. Furthermore, we examined the swimming behavior of the fliG mutants lacking CheY. The results suggested that the conformation of FliG in E144D mutant was similar to that in the wild type. However, that of G214S and G215A caused a steric hindrance in FliG. The conformational change in FliGM triggered by binding CheY may lead to a rapid change of direction and may occur in both directional states.
The bacterial flagellar motor is a unique supramolecular complex which converts ion flow into rotational force. Many biological devices mainly use two types of ions, proton and sodium ion. This is probably because of the fact that life originated in seawater, which is rich in protons and sodium ions. The polar flagellar motor in Vibrio is coupled with sodium ion and the energy converting unit of the motor is composed of two membrane proteins, PomA and PomB. It has been shown that the ion binding residue essential for ion transduction is the conserved aspartic acid residue (PomB-D24) in the PomB transmembrane region. To reveal the mechanism of ion selectivity, we identified essential residues, PomA-T158 and PomA-T186, other than PomB-D24, in the Na + -driven flagellar motor. It has been shown that the side chain of threonine contacts Na + in Na + -coupled transporters. We monitored the Na + -binding specific structural changes using ATR-FTIR spectroscopy. The signals were abolished in PomA-T158A and -T186A, as well as in PomB-D24N. Molecular dynamics simulations further confirmed the strong binding of Na + to D24 and showed that T158A and T186A hindered the Na + binding and transportation. The data indicate that two threonine residues (PomA-T158 and PomA-T186), together with PomB-D24, are important for Na + conduction in the Vibrio flagellar motor. The results contribute to clarify the mechanism of ion recognition and conversion of ion flow into mechanical force.
Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator.
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