Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition
Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition
复制标题
DOI：
10.1074/jbc.m400993200
复制
发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Jerala, R
中科院分区：
文献类型：
--
作者：
Gruber, A;Mancek, M;Jerala, R
由MD-2和Toll样受体4（TLR 4）的胞外结构域缔合产生的受体复合物介导脂多糖（LPS）跨细胞膜的信号转导。我们准备了一个三级结构模型的MD-2，同源的脂质结合蛋白的已知结构的基础上。对纯化的细菌表达的MD-2的圆二色性光谱的分析表明高含量的β-型二级结构，与结构模型一致。细菌表达的MD-2能够赋予LPS对表达TLR 4的细胞的反应性，尽管缺乏糖基化。我们确定了几个集群的MD-2的表面上的碱性残基。包含残基Lys（89）-Arg（90）-Lys（91）和Lys（125）-Lys（125）的两个簇中的每一个的突变在与TLR 4共表达时或在作为可溶性蛋白质添加到过表达TLR 4的细胞的上清液中时显著降低相应MD-2突变体的信号转导。这些基本簇位于β-折叠夹层的边缘，其在与尼曼-匹克病C2（NPC 2）、尘螨过敏原Der p2和神经节苷脂GM 2激活蛋白连接的胆固醇结合蛋白中形成疏水口袋。相比之下，根据该模型，由Arg（69）-Lys（72）组成的另一个基本簇的突变远离疏水口袋，仅微弱地降低MD-2活性。此外，通过模型预测，添加肽（包括Cys（95）和Cys（105）之间的表面环），特别是以氧化形式，分别在应用于单核细胞和成纤维细胞时降低LPS诱导的肿瘤坏死因子α和白细胞介素-8的产生，支持其参与LPS信号传导。我们的MD-2的结构模型得到了生化分析的证实，并有助于解开LPS识别中的分子相互作用。
The receptor complex resulting from association of MD-2 and the ectodomain of Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signal transduction across the cell membrane. We prepared a tertiary structure model of MD-2, based on the known structures of homologous lipid-binding proteins. Analysis of circular dichroic spectra of purified bacterially expressed MD-2 indicates high content of beta-type secondary structure, in agreement with the structural model. Bacterially expressed MD-2 was able to confer LPS responsiveness to cells expressing TLR4 despite lacking glycosylation. We identified several clusters of basic residues on the surface of MD-2. Mutation of each of two clusters encompassing the residues Lys(89)-Arg(90)-Lys(91) and Lys(125)-Lys(125) significantly decreased the signal transduction of the respective MD-2 mutants either upon co-expression with TLR4 or upon addition as soluble protein into the supernatant of cells overexpressing TLR4. These basic clusters lie at the edge of the beta-sheet sandwich, which in cholesterol-binding protein connected to Niemann-Pick disease C2 (NPC2), dust mite allergen Der p2, and ganglioside GM2-activator protein form a hydrophobic pocket. In contrast, mutation of another basic cluster composed of Arg(69)-Lys(72), which according to the model lies further apart from the hydrophobic pocket only weakly decreased MD-2 activity. Furthermore, addition of the peptide, comprising the surface loop between Cys(95) and Cys(105), predicted by model, particularly in oxidized form, decreased LPS-induced production of tumor necrosis factor alpha and interleukin-8 upon application to monocytic cells and fibroblasts, respectively, supporting its involvement in LPS signaling. Our structural model of MD-2 is corroborated by biochemical analysis and contributes to the unraveling of molecular interactions in LPS recognition.