Yasuhiro Onoue

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.

A giant liposome for single-molecule observation of conformational changes in membrane proteins

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F(O)F(1)) in liposomes tens of mum in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.

Effect of FliG three amino acids deletion in Vibrio polar-flagellar rotation and formation.

Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (ΔPSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(ΔPSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium

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.

Chemo-Mechanical Coupling in the Rotary Molecular Motor F1-ATPase

F1-ATPase is a molecular motor in which the central γ subunit rotates inside the cylinder made of α3β3 subunits. The rotation is powered by ATP hydrolysis in three catalytic sites, and reverse rotation of the γ subunit by an external force leads to ATP synthesis in the catalytic sites. Single-molecule studies have revealed how the mechanical rotation is coupled to the chemical reactions in the three catalytic sites: binding/release of ATP, ADP, and phosphate, and hydrolysis/synthesis of ATP.

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliGC). We have identified point mutations in FliGC from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliGC from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliGC are observed in the Cα1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliGC at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix α1 in the Cα1-6 domain of FliGC contribute to ensure the precise regulation of the motor switching.

Serine suppresses the motor function of a periplasmic PomB mutation in the Vibrio flagella stator.

The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross‐linking experiments of double‐Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double‐Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)‐biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che− mutants, in either a CCW‐locked or clockwise (CW)‐locked strain. In contrast, in a ΔcheY (CCW‐locked) strain, Vibrio (L160C/I186C) required serine to be rescued. We speculate that CheY affects stator conformation and motility restoration by serine is independent on the chemotaxis signaling pathway.

Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli.

The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB91, PotB129, and PotB138, with various chimeric junctions. When those chimeric proteins were produced with PomA in a ΔmotAB strain of E. coli or in ΔpomAB and ΔpomAB ΔmotX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the T ring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in ΔpomAB, PotB138 had the highest motility. The difference between the origin of the α1 and α2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.

Stimulation of F1-ATPase activity by sodium dodecyl sulfate

F(1)-ATPase is a rotary molecular motor in which the gamma subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F(1)-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3-4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-mum bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F(1) molecules into an active form but it does not alter or enhance the function of already active F(1) molecules significantly.

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.