S. Nagashima

Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling

The bacterial flagellar filament is a helical propeller constructed from 11 protofilaments of a single protein, flagellin. The filament switches between left- and right-handed supercoiled forms when bacteria switch their swimming mode between running and tumbling. Supercoiling is produced by two different packing interactions of flagellin called L and R. In switching from L to R, the intersubunit distance (∼52 Å) along the protofilament decreases by 0.8 Å. Changes in the number of L and R protofilaments govern supercoiling of the filament. Here we report the 2.0 Å resolution crystal structure of a Salmonella flagellin fragment of relative molecular mass 41,300. The crystal contains pairs of antiparallel straight protofilaments with the R-type repeat. By simulated extension of the protofilament model, we have identified possible switch regions responsible for the bi-stable mechanical switch that generates the 0.8 Å difference in repeat distance.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Crystallization of a core fragment of the flagellar hook protein FlgE

A core fragment of the bacterial flagellar hook protein FlgE was overexpressed, purified and crystallized. The crystal diffracted to 1.6 A resolution using synchrotron X-radiation. The crystal belongs to the orthorhombic crystal system, with space group P2(1)2(1)2 and unit-cell parameters a = 128.4, b = 48.8, c = 96.7 A. SeMet protein was also overexpressed, purified, crystallized and a set of 2.3 A MAD data was collected.

Crystallization and preliminary X-ray analysis of FlgA, a periplasmic protein essential for flagellar P-ring assembly

Salmonella FlgA, a periplasmic protein essential for flagellar P-ring assembly, has been crystallized in two forms. The native protein crystallized in space group C222, with unit-cell parameters a = 107.5, b = 131.8, c = 49.4 Å, and diffracted to about 2.0 Å resolution (crystal form I). In this crystal, the asymmetric unit is likely to contain one molecule, with a solvent content of 66.8%. Selenomethionine-labelled FlgA protein crystallized in space group C222(1), with unit-cell parameters a = 53.2, b = 162.5, c = 103.5 Å, and diffracted to 2.7 Å resolution (crystal form II). In crystal form II, the asymmetric unit contained two molecules with a solvent content of 48.0%. The multiple-wavelength and single-wavelength anomalous dispersion methods allowed the visualization of the electron-density distributions of the form I and II crystals, respectively. The two maps suggested that FlgA is in two different conformations in the two crystals.