Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA.
Biochemistry. 2010 Aug 31;49(34):7403-10. doi: 10.1021/bi100887w.
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.
炭疽毒素受体 1（ANTXR1）/肿瘤内皮标记物 8（TEM8）是两种已知的蛋白细胞表面炭疽毒素受体之一。存在于 ANTXR1 的整联蛋白样插入（I）结构域中的金属离子依赖性粘附位点（MIDAS）介导炭疽毒素亚单位保护性抗原（PA）的结合。在这里，我们提供的证据表明，I 结构域中的单点突变可以覆盖细胞内信号介导的 ANTXR1 配体结合活性的调节。先前报道的 ANTXR1 的 MIDAS 突变体（T118A）被发现保留了正常的金属离子结合和二级结构，但不能结合 PA，与锁定的非活性状态一致。相反，将保守的 I 结构域苯丙氨酸残基突变为色氨酸（F205W）增加了与 PA 结合的细胞表面 ANTXR1 的比例，与锁定的活性状态一致。有趣的是，通过分离的 ANTXR1 I 结构域结合的 PA 的 K(D)和总量不受 F205W 突变的影响，表明在不存在内向外信号的情况下，ANTXR1 优先处于活性状态。圆二色性（CD）光谱和（1）H-（15）N 异核单量子相干（HSQC）核磁共振（NMR）表明，尽管 T118A、F205W 和 WT I 结构域之间的毒素结合能力差异超过 10(3)倍，但它们之间的结构变化很小。毒素结合的调节对毒素抑制剂的设计和针对 ANTXR1 的抗肿瘤治疗具有重要意义。
