Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
J Biol Chem. 2012 Nov 30;287(49):41089-102. doi: 10.1074/jbc.M112.412809. Epub 2012 Sep 19.
We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD(+) with high affinity (K(D) = 52.3 ± 12.2 μM). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K(D) = 1.7 ± 0.2 μM, K(i) = 1.8 ± 0.4 μM) and PJ34 (K(D) = 5.8 ± 2.6 μM, K(i) = 9.6 ± 0.3 μM)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD(+) binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.
我们从致病性 G9241 株蜡样芽胞杆菌中鉴定出了 Certhrax,这是一种首例炭疽样 mART 毒素。Certhrax 与炭疽杆菌的炭疽致死因子有 31%的序列同一性;然而,我们已经表明,Certhrax 的毒性存在于 mART 结构域中,而炭疽则使用金属蛋白酶机制。像炭疽致死因子一样,Certhrax 被发现需要保护性抗原才能进入宿主细胞。这种双结构域酶对哺乳动物细胞的毒性比炭疽致死因子高 60 倍。Certhrax 在感染后 10 分钟内在小鼠 RAW264.7 细胞内定位于不同区域,并且在其细胞位置上是核外的。催化残基的取代表明 mART 功能负责毒性,并且它与 NAD(+) 具有高亲和力(K(D) = 52.3 ± 12.2 μM)。我们报告了 Certhrax 的 2.2 Å 结构,突出了其与炭疽致死因子的结构相似性和差异性。我们还确定了两种良好抑制剂(P6(K(D) = 1.7 ± 0.2 μM,K(i) = 1.8 ± 0.4 μM)和 PJ34(K(D) = 5.8 ± 2.6 μM,K(i) = 9.6 ± 0.3 μM))与 Certhrax 复合物的晶体结构。与该家族中的其他毒素一样,带磷酸的烟酰胺环随结合抑制剂向 NAD(+) 结合位点移动。这些结果表明 Certhrax 可能在蜡样芽胞杆菌的发病机制中很重要。