Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

Abstract

IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

Figure 1

(a) Monoclinic crystals of IpaD crystal form 2 produced by in-drop proteolysis. (b) SDS–PAGE of protein derived from crystals with (CF-3 and CF-5) and without (CF-1) subtilisin treatment (see text for full description). The subscript denotes the portion of IpaD present in each band as determined by sequencing (FL, full length; Δ120, N-terminal deletion to residue 120). Molecular-weight markers are shown in lane 2; the marker identified with ‘M’ corresponds to a MW of 42 kDa.

Figure 2

Harker section v = 0.5 of the native Patterson map of IpaD crystal form 1 calculated using PATTERSON (Collaborative Computational Project, Number 4, 1994 ▶) at 3.5 Å. The map is drawn with a minimum contour level of 1.5σ with 0.5σ increments.

Figure 3

The κ = 180° section of the self-rotation function calculated for the crystal form 4 SeMet peak 1 data set using MOLREP (Collaborative Computational Project, Number 4, 1994 ▶) with an integration radius of 20.0 Å and data in the resolution range 15–4 Å. The peak (marked with an X) at (ω, ϕ) = (22, 0°) represents 91% of the peak for the crystallographic twofold axis.