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.

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na+-driven flagellar motor.

Many bacteria move using their flagellar motor, which generates torque through the interaction between the stator and rotor. The most important component of the rotor for torque generation is FliG. FliG consists of three domains: FliGN, FliGM, and FliGC. FliGC contains a site(s) that interacts with the stator. In this study, we examined the physical properties of three FliG constructs, FliGFull, FliGMC, and FliGC, derived from sodium-driven polar flagella of marine Vibrio. Size exclusion chromatography revealed that FliG changes conformational states under two different pH conditions. Circular dichroism spectroscopy also revealed that the contents of α-helices in FliG slightly changed under these pH conditions. Furthermore, we examined the thermal stability of the FliG constructs using differential scanning calorimetry. Based on the results, we speculate that each domain of FliG denatures independently. This study provides basic information on the biophysical characteristics of FliG, a component of the flagellar motor.

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.

Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor.

The membrane motor proteins, PomA (polar flagellar motility protein A) and PomB (polar flagellar motility protein B), of Vibrio alginolyticus form a stator complex that converts energy from the ion flow to mechanical work in bacterial flagellar motors. The cytoplasmic domain of PomA is believed to interact with the rotor protein FliG to make a torque. In this study, to investigate the function of the cytoplasmic domain of PomA, we constructed a series of fragments that flank the cytoplasmic loop of PomA between the second and third transmembrane (TM) domains (A-loop) and the C-terminal region, and expressed them in Escherichia coli together with PomA and PotB (a chimeric protein of PomB and MotB). We observed a dominant-negative effect of one PomA fragment on motility. We confirmed that these PomA fragments localized both in the membrane fraction and in the cytoplasmic fraction, and induced bacterial growth delay. Effect of additional point and deletion mutations into this fragment implies that the C-terminal region and TM domains used as a linker play a significant part in these observations. From these results, we conclude that the PomA fragments retain the structure important for functions. We expect that further constructions will provide a variety of experimental approaches to characterize the interaction between PomA and FliG.

Biophysical characterization of the C-terminal region of FliG, an essential rotor component of the Na+-driven flagellar motor.

The bacterial flagellar motor generates a rotational force by the flow of ions through the membrane. The rotational force is generated by the interaction between the cytoplasmic regions of the rotor and the stator. FliG is directly involved in the torque generation of the rotor protein by its interaction. FliG is composed of three domains: the N-terminal, Middle and C-terminal domains, based on its structure. The C-terminal domain of FliG is assumed to be important for the interaction with the stator that generates torque. In this study, using CD spectra, gel filtration chromatography and DSC (differential scanning calorimetry), we characterized the physical properties of the C-terminal domain (G214-Stop) of wild-type (WT) FliG and its non-motile phenotype mutant derivatives (L259Q, L270R and L271P), which were derived from the sodium-driven motor of Vibrio. The CD spectra and gel filtration chromatography revealed a slight difference between the WT and the mutant FliG proteins, but the DSC results suggested a large difference in their stabilities. That structural difference was confirmed by differences in protease sensitivity. Based on these results, we conclude that mutations which confer the non-motile phenotype destabilize the C-terminal domain of FliG.

Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na(+) driven flagellar motor.

Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na(+) influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function.