Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding

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• 作者：Jordan D. Ramey ; Valerie A. Villareal ; Charles Ng ; Sabrina C. Ward ; Jian-Ping Xiong ; Robert T. Clubb ; Kenneth A. Bradley
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：August 31, 2010
• 年：2010
• 卷：49
• 期：34
• 页码：7403-7410
• 全文大小：869K
• 年卷期：v.49,no.34(August 31, 2010)
• ISSN：1520-4995

文摘

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 ¹H−¹⁵N 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 10³-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.