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

Journal article

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.

Authors: Johnson, S; Roversi, P; Espina, M; Deane, J; Birket, S

• Publication status: Published
• Journal: Acta crystallographica. Section F, Structural biology and crystallization communications
• Volume: 62
• Issue: Pt 9
• Pages: 865-868
• Publication date: 2006-09-01
• DOI: 10.1107/s1744309106027047
• EISSN: 1744-3091
• ISSN: 1744-3091
• Language: English
• Keywords: Crystallization; Bacterial Proteins; Crystallography, X-Ray; Hydrolysis; Antigens, Bacterial; Gene Expression Regulation, Bacterial; Shigella flexneri; Peptide Hydrolases