Katona Gergely, Berglund Gunnar I, Hajdu Janos, Gráf László, Szilágyi László
Department of Biochemistry, Eötvös Loránd University, Puskin u. 3., Budapest, 1088, Hungary.
J Mol Biol. 2002 Feb 1;315(5):1209-18. doi: 10.1006/jmbi.2001.5305.
Severe neurodegradative brain diseases, like Alzheimer, are tightly linked with proteolytic activity in the human brain. Proteinases expressed in the brain, such as human trypsin IV, are likely to be involved in the pathomechanism of these diseases. The observation of amyloid formed in the brain of transgenic mice expressing human trypsin IV supports this hypothesis. Human trypsin IV is also resistant towards all studied naturally occurring polypeptide inhibitors. It has been postulated that the substitution of Gly193 to arginine is responsible for this inhibitor resistance. Here we report the X-ray structure of human trypsin IV in complex with the inhibitor benzamidine at 1.7 A resolution. The overall fold of human trypsin IV is similar to human trypsin I, with a root-mean square deviation of only 0.5 A for all C(alpha) positions. The crystal structure reveals the orientation of the side-chain of Arg193, which occupies an extended conformation and fills the S2' subsite. An analysis of surface electrostatic potentials shows an unusually strong clustering of positive charges around the primary specificity pocket, to which the side-chain of Arg193 also contributes. These unique features of the crystal structure provide a structural basis for the enhanced inhibitor resistance, and enhanced substrate restriction, of human trypsin IV.
严重的神经退行性脑部疾病，如阿尔茨海默病，与人类大脑中的蛋白水解活性紧密相关。大脑中表达的蛋白酶，如人胰蛋白酶IV，可能参与了这些疾病的发病机制。在表达人胰蛋白酶IV的转基因小鼠大脑中形成淀粉样蛋白的观察结果支持了这一假设。人胰蛋白酶IV对所有研究过的天然存在的多肽抑制剂也具有抗性。据推测，甘氨酸193被精氨酸取代是导致这种抑制剂抗性的原因。在此，我们报告了人胰蛋白酶IV与抑制剂苯甲脒复合物在1.7埃分辨率下的X射线结构。人胰蛋白酶IV的整体折叠与人类胰蛋白酶I相似，所有Cα位置的均方根偏差仅为0.5埃。晶体结构揭示了精氨酸193侧链的取向，其占据伸展构象并填充S2'亚位点。表面静电势分析表明，在主要特异性口袋周围存在异常强烈的正电荷聚集，精氨酸193的侧链也对其有贡献。晶体结构的这些独特特征为人类胰蛋白酶IV增强的抑制剂抗性和增强的底物特异性提供了结构基础。
