Mapping the anthrax protective antigen binding site on the lethal and edema factors
Mapping the anthrax protective antigen binding site on the lethal and edema factors
复制标题
DOI：
10.1074/jbc.m109997200
复制
发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Collier, RJ
中科院分区：
文献类型：
--
作者：
Lacy, DB;Mourez, M;Collier, RJ
炭疽水肿因子(EF)和致死因子(LF)进入真核细胞胞浆的能力取决于它们在炭疽保护性抗原(PA)存在下跨内体膜转运的能力。在这里，我们报告了EF和LF的N-末端结构域(分别为EFN和LFN)的属性，这些属性对它们与PA的初始相互作用是至关重要的。我们发现，LFN前36个残基的缺失对其与PA的结合或转位能力没有影响。为了定位PA的结合位点，我们利用Lf的三维结构和EF与Lf之间的序列相似性来选择突变位置。我们在LFN中发现了7个位点(Asp-182、Asp-187、Leu-188、Tyr-223、His-229、Leu-235和Tyr-236)，其中Ala的突变产生了显著的结合缺陷，其中H229A和Y236A几乎完全消除了结合。EFN的同源突变体显示出几乎相同的缺陷。细胞毒性实验证实，LFN突变对中毒有影响。这7个突变敏感氨基酸聚集在Lf表面，形成一个既疏水又亲水的小卷曲斑块。我们建议这个补丁构成PA的识别站点。
Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EFN and LFN, respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LFN had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LFN (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EFN displayed nearly identical defects. Cytotoxicity assays confirmed that the LFN mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.