title: The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function.
abstract: Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1-171, contains two amphipathic domains spanning residues 24-41 and 66-83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His₆-EspD₁₋₁₇₁ adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His₆-EspD₁₋₁₇₁ undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp₄₇ into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His₆-EspD₁₋₁₇₁ forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional.
authors.affiliation: Institute of Parasitology and Centre for Host-Parasite Interactions, Macdonald Campus of McGill University, Ste-Anne-de-Bellevue, Québec H9X3V9, Canada.
authors.forename: Dayal
authors.initials: D
authors.lastname: Dasanayake
authors.forename: Manon
authors.initials: M
authors.lastname: Richaud
authors.forename: Normand
authors.initials: N
authors.lastname: Cyr
authors.forename: Celia
authors.initials: C
authors.lastname: Caballero-Franco
authors.forename: Sabrina
authors.initials: S
authors.lastname: Pittroff
authors.forename: Ron M
authors.initials: RM
authors.lastname: Finn
authors.forename: Juan
authors.initials: J
authors.lastname: Ausió
authors.forename: Wensheng
authors.initials: W
authors.lastname: Luo
authors.forename: Michael S
authors.initials: MS
authors.lastname: Donnenberg
authors.forename: Armando
authors.initials: A
authors.lastname: Jardim
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D054324:Enterohemorrhagic Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D016415:Sequence Alignment; D013057:Spectrum Analysis
chemical_list: D029968:Escherichia coli Proteins; C474481:EspD protein, E coli; D026901:Membrane Transport Proteins; D010718:Phosphatidylserines