A partial atomic structure for the flagellar hook of Salmonella typhimurium

Abstract

The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The N- and C-terminal sequences of the eight axial proteins were predicted to form interlocking alpha-domains generating an axial tube. We report on an approximately 1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an approximately 2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.

Fig. 1.

EM and image analysis of the hook. The average of all of the hook data sets seen as a projection of the three-dimensional map is shown in Left. Such a projection is equivalent to the average hook image as it would be seen in the electron microscope. (Scale bar: 10 nm.) A display of the layer-line data [G_n(R, Z)] for the data included in the average is shown in Right. The logarithm of the amplitude is displayed to make the weaker layer lines visible. The cutoff in resolution is 0.9 nm.

Fig. 2.

Three-dimensional maps of the hook. (a) A section taken perpendicular to the helical axis. (b) A cylindrical section through the wall of the central tube. The rod-like features lining the central putative protein channel are spaced about 1 nm apart in the azimuthal (horizontal) direction. (c) A vector diagram showing the directions that the 5, 11, 6, and 1 start helices in b. (d) A surface representation of the hook density map. (e) A surface representation with the front half of the structure removed to reveal domain D0. The three domains of the hook subunit are labeled D0, D1, and D2. (Scale bar: 10 nm.)

Fig. 3.

The atomic model of the outer two domains of the hook. (a) The structure of a major fragment of the hook subunit as determined by x-ray crystallography (28). β-Sheet is shown in yellow and α-helical segments in red. The lower domain (A) contains both N- and C-terminal regions, has the more evolutionarily conserved amino acid sequence, and corresponds to domain D1. The upper domain (B) corresponds to D2. (b) The change in domain arrangement involved in the docking and refinement. Three models are superimposed by fitting the D1 domains to one another. The figure shows the difference in the angle between domains D1 and D2 after refinement. The crystal structure model is shown in blue; the model refined against the C. crescentus map, green; the model refined against the S. typhimurium map, red. (c) A stereo pair of the atomic structure docked into the S. typhimurium map. Some of the subunits are shown outside the map. (d) A stereo pair showing the atomic structure docked into the C. crescentus map.

Fig. 4.

A comparison of the sequences of hook proteins. (a) Shown are the sequences corresponding to the proteins FlgE of S. typhimurium (upper sequence) and C. crescentus (lower sequence). The residues corresponding to domain D1 (or A in Fig. 3a) are shown in yellow, and those corresponding to domain D2 (or B in Fig. 3a) are shown in blue. The large insert found in C. crescentus but not S. typhimurium is shown in magenta. The other four inserts mapped in b and c are shown in green. Below the sequences is a bar graph showing sequence identity in FlgE for 13 bacterial species. (The height of the tallest bar corresponds to 13.) Note that the sequence assigned to domain D2 has more inserts than that of D1. (b) Space-filling model of hook subunits as seen from the outside. The sites of the small inserts are shown in green, and the site of the large insert in the hook of C. crescentus is shown in magenta. The yellow and blue portions correspond to domains D1 and D2, respectively, and the yellow and blue sequences in a.(c) Space-filling model of the hook subunits as seen from the inside the hook. The N and C termini, shown in blue and red, respectively, lie close to one another. The axis of the hook is vertical with the cell-proximal side at the bottom. (d) A stereo pair showing a surface representation of the difference map in which the S. typhimurium map is subtracted from the C. crescentus map. The atomic model derived by docking is shown with the site of the insert in C. crescentus marked by a magenta rod.