Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore.
Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
Sci Rep. 2017 Mar 28;7:45230. doi: 10.1038/srep45230.
Six-domain gelsolin regulates actin structural dynamics through its abilities to sever, cap and uncap F-actin. These activities are modulated by various cellular parameters like Ca and pH. Until now, only the molecular activation mechanism of gelsolin by Ca has been understood relatively well. The fragment comprising the first domain and six residues from the linker region into the second domain has been shown to be similar to the full-length protein in F-actin severing activity in the absence of Ca at pH 5. To understand how this gelsolin fragment is activated for F-actin severing by lowering pH, we solved its NMR structures at both pH 7.3 and 5 in the absence of Ca and measured the pKa values of acidic amino acid residues and histidine residues. The overall structure and dynamics of the fragment are not affected significantly by pH. Nevertheless, local structural changes caused by protonation of His29 and Asp109 result in the activation on lowering the pH, and protonation of His151 directly effects filament binding since it resides in the gelsolin/actin interface. Mutagenesis studies support that His29, Asp109 and His151 play important roles in the pH-dependent severing activity of the gelsolin fragment.
六域凝胶蛋白通过其切断、封端和去封端 F-肌动蛋白的能力来调节肌动蛋白的结构动态。这些活性受到 Ca 和 pH 等各种细胞参数的调节。到目前为止，人们相对较好地理解了 Ca 对凝胶蛋白的分子激活机制。包含第一个结构域和连接区的六个残基到第二个结构域的片段在没有 Ca 的情况下，在 pH 5 下与全长蛋白的 F-肌动蛋白切断活性相似。为了了解该凝胶蛋白片段如何通过降低 pH 值被激活以进行 F-肌动蛋白切断，我们在没有 Ca 的情况下分别在 pH 7.3 和 5 下解决了其 NMR 结构，并测量了酸性氨基酸残基和组氨酸残基的 pKa 值。片段的整体结构和动力学不受 pH 值的显著影响。然而，由于 His29 和 Asp109 的质子化引起的局部结构变化导致 pH 值降低时的激活，并且 His151 直接影响丝状结合，因为它位于凝胶蛋白/肌动蛋白界面中。突变研究表明，His29、Asp109 和 His151 在凝胶蛋白片段的 pH 依赖性切断活性中起重要作用。
