Polverino de Laureto Patrizia, Taddei Niccolò, Frare Erica, Capanni Cristina, Costantini Silvia, Zurdo Jesús, Chiti Fabrizio, Dobson Christopher M, Fontana Angelo
CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121, Padua, Italy.
J Mol Biol. 2003 Nov 14;334(1):129-41. doi: 10.1016/j.jmb.2003.09.024.
The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.
SH3结构域是由60 - 85个氨基酸残基组成的小蛋白质模块，存在于许多参与细胞内信号转导的蛋白质中。牛磷脂酰肌醇3'-激酶（PI3 - SH3）的p85α亚基的SH3结构域在酸性溶液中会形成一种紧密的变性状态，由此很容易形成淀粉样纤维。已发现这种聚集过程会受到溶液条件的显著调节。在这里，我们分别使用蛋白酶K和胃蛋白酶通过有限蛋白酶解实验分析了PI3 - SH3天然态和酸变性态的构象特征。此外，我们分析了PI3 - SH3在pH 1.2和2.0的聚集和淀粉样形成过程中不同阶段被胃蛋白酶水解的倾向，并将这些条件下的蛋白酶解位点与天然态和聚集态PI3 - SH3的构象特征进行了比较。结果表明，在低pH下形成的PI3 - SH3变性态对蛋白酶解相对抗性较强，表明它是部分折叠的。天然蛋白质中连接β链b和c的长环是该结构中最易被蛋白酶解的区域。值得注意的是，最初在酸性溶液中由这种变性态形成的PI3 - SH3聚集体对长环的蛋白酶解敏感性增强，这表明该蛋白质在聚集早期变得更加展开。相比之下，长时间形成的更规则的淀粉样纤维对蛋白酶解完全抗性。我们认为，最初形成的蛋白质聚集体是相对动态的物种，能够很容易地重新组织它们的相互作用，以形成非常有序的纤维状结构。此外，早期聚集体中多肽链的无序和非天然特性可能对决定这些物种在先前研究中所揭示的高细胞毒性很重要。
