title: Anthrax toxin receptor 1/tumor endothelial marker 8: mutation of conserved inserted domain residues overrides cytosolic control of protective antigen binding.
abstract: Anthrax toxin receptor 1 (ANTXR1)/tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the K(D) and total amount of PA bound by the isolated ANTXR1 I domain were not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and (1)H-(15)N heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W, and WT I domains were minor despite a greater than 10(3)-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for antitumor therapies.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA.
authors.forename: Jordan D
authors.initials: JD
authors.lastname: Ramey
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Villareal
authors.forename: Charles
authors.initials: C
authors.lastname: Ng
authors.forename: Sabrina C
authors.initials: SC
authors.lastname: Ward
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Xiong
authors.forename: Robert T
authors.initials: RT
authors.lastname: Clubb
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Bradley
mesh_terms: D000818:Animals; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D052250:Nitric Oxide Synthase Type III; D017433:Protein Structure, Secondary / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide
chemical_list: D000941:Antigens; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D014408:Biomarkers, Tumor; D008565:Membrane Proteins; D011506:Proteins; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III