Abstract
A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH(CDelta5) and diffraction data were collected to 1.9 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 A, beta = 96.5 degrees. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 A3 Da(-1) for two molecules per asymmetric unit, corresponding to a solvent content of 33%.
Highlights
Type III secretion systems (TTSSs) are essential virulence determinants in many Gram-negative bacterial pathogens
The type III secretion system (TTSS) consists of a ‘needle complex’ composed of an external hollow needle held within a basal body that traverses both bacterial membranes
The flagellum is a helical superstructure that is assembled by the export of the flagellar components through a central channel of the flagellum in a process highly analogous to the initial steps of export through a virulence TTSS (Mimori et al, 1995; Yonekura et al, 2003)
Summary
Type III secretion systems (TTSSs) are essential virulence determinants in many Gram-negative bacterial pathogens. The Shigella flexneri needle is composed of the $9 kDa protein MxiH that assembles in a helical superstructure architecturally similar to the flagellar hook and filament (Cordes et al, 2003; Samatey, Matsunami, Imada, Nagashima, Shaikh et al, 2004; Yonekura et al, 2003). A model of the innermost (D0) domain of flagellin built in a high-resolution EM map forms a tube $70 Ain diameter with a central channel of $20 A , similar to the dimensions described for the S. flexneri needle (Yonekura et al, 2003; Cordes et al, 2003) Despite their different sizes and lack of sequence homology, these proteins may be structurally similar in the regions used to pack into superhelices. Largely owing to the small size of MxiH (9 kDa), prevention of polymerization was achieved by the removal of five C-terminal residues (MxiHCÁ5; Kenjale et al, 2005)
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