title: Self-association of the transmembrane domain of an anthrax toxin receptor.
abstract: Protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted individually by Bacillus anthracis. These components of anthrax toxin must then assemble into complexes to intoxicate mammalian cells. Toxin assembly initiates when molecules of PA bind mammalian receptors ANTXR1/2 and are cleaved by surface proteases into 20 kDa and 63 kDa fragments. After PA20 dissociates, receptor-bound PA63 homo-oligomerizes into heptamers. Oligomeric PA63 binds EF and LF and these complexes are internalized into an acidic compartment where the two enzymatic components are translocated across the membrane by a channel formed by heptameric PA63. Since oligomerization of PA63 is required to bind and translocate the enzymatic components, we sought to determine whether interactions between toxin receptors could facilitate the assembly process. In the present work, we performed a co-immunoprecipitation experiment to demonstrate that ANTXR1 is oligomeric in mammalian cells. Computer modeling predicted the self-association of the ANTXR1 transmembrane domain and we detected oligomerization of ANTXR1 transmembrane domain peptides in the membrane-mimetic environment of SDS micelles using fluorescence resonance energy transfer. Furthermore, the ANTXR1 transmembrane domain mediated oligomerization of a reporter protein construct in a bacterial membrane. In both assays, mutations that disrupted the interaction were consistent with the interaction being mediated through an asymmetric binding interface. Mutations that impaired self-association of the transmembrane domain reduced the rate of PA63 heptamer formation on the mammalian cell surface. Our findings indicate that ANTXR1 transmembrane domains self-associate and that these interactions may stabilize intermediate oligomerization states of ANTXR1-PA63 complexes.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8.
authors.forename: Mandy Y
authors.initials: MY
authors.lastname: Go
authors.forename: Sanguk
authors.initials: S
authors.lastname: Kim
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Partridge
authors.forename: Roman A
authors.initials: RA
authors.lastname: Melnyk
authors.forename: Arianna
authors.initials: A
authors.lastname: Rath
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
authors.forename: Jeremy
authors.initials: J
authors.lastname: Mogridge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000881:Anthrax / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry*; D001408:Bacillus anthracis / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin