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J. Biol. Chem., Vol. 282, Issue 44, 32144-32151, November 2, 2007

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Identification of the MxiH Needle Protein Residues Responsible for Anchoring Invasion Plasmid Antigen D to the Type III Secretion Needle Tip^*

From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus to serve as a conduit for injecting host-altering effector proteins into the membrane and cytoplasm of the targeted cell. The type III secretion apparatus is composed of a basal body and an exposed needle that is an extended polymer of MxiH with a 2.0-nm inner channel. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control type III secretion. The atomic structures of MxiH and IpaD have been solved. MxiH (8.3 kDa) is a helix-turn-helix, whereas IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. These structures alone, however, have not been sufficient to produce a workable in silico model by which IpaD docks at the needle tip. Thus, the work presented here provides an initial step in understanding this important protein-protein interaction. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of Asn-43, Leu-47, and Tyr-50 residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype of S. flexneri. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly.

Received for publication, April 24, 2007 , and in revised form, September 5, 2007.

The chemical shift assignments for MxiH^C5 have been deposited at the Biological Magnetic Resonance Bank (BMRB accession number 15214).

^* This work was supported by Public Health Service Grants AI034428 and AI057927 (to W. D. P.), AI067858 (to W. L. P.), and RR017708 (CoBRE Protein Structure and Function Program) and Kansas University start-up funds (to R. N. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045-7534. Tel.: 785-864-3299; Fax: 785-864-5294; E-mail: pickingw{at}ku.edu.

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