title: Identification of the MxiH needle protein residues responsible for anchoring invasion plasmid antigen D to the type III secretion needle tip.
abstract: 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.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: Lingling
authors.initials: L
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Olive
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Smith
authors.forename: William D
authors.initials: WD
authors.lastname: Picking
authors.forename: Roberto N
authors.initials: RN
authors.lastname: De Guzman
authors.forename: Wendy L
authors.initials: WL
authors.lastname: Picking
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012763:Shigella flexneri / Q000737:chemistry / Q000378:metabolism*