Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India.
Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India.
Biosci Rep. 2018 Oct 5;38(5). doi: 10.1042/BSR20181072. Print 2018 Oct 31.
Serine protease high temperature requirement protease A2 (HtrA2) is involved in apoptosis and protein quality control. However, one of its murine inactive mutants (S276C aka mnd2) is associated with motor neuron degeneration 2. Similarly, this conserved mutation in human HtrA2 (hHtrA2) also renders the protease inactive, implicating pathogenicity. However, the structural determinants for its inactivation have not yet been elucidated. Here, using multidisciplinary approach, we studied the structural basis of inactivity associated with this mutation in hHtrA2. Characterization of secondary and tertiary structural properties, protein stability, oligomeric properties, and enzyme activity for both wild-type and mutant has been performed using biophysical and functional enzymology studies. The structural comparison at atomic resolution has been carried out using X-ray crystallography. While enzyme kinetics showed inactivity, spectroscopic probes did not identify any significant secondary structural changes in the mutant. X-ray crystallographic analysis of the mutant protein at 2 Å resolution highlighted the significance of a water molecule that plays important role in mediating intermolecular interactions for maintaining the functional ensemble of the protease. Overall, the crystallographic data along with biophysical and enzymology studies helped decipher the structural basis of inactivity of hHtrA2S276C, which might pave way toward further investigating its correlation with aberration of normal cellular functions, hence pathogenicity.
丝氨酸蛋白酶高温需求蛋白酶 A2（HtrA2）参与细胞凋亡和蛋白质质量控制。然而，其一种鼠类无活性突变体（S276C，又名 mnd2）与运动神经元退行性变 2 型有关。同样，人类 HtrA2 中的这种保守突变（hHtrA2）也使蛋白酶失活，暗示其具有致病性。然而，其失活的结构决定因素尚未阐明。在这里，我们使用多学科方法研究了与 hHtrA2 中这种突变相关的无活性的结构基础。使用生物物理和功能酶学研究对野生型和突变型的二级和三级结构特性、蛋白质稳定性、寡聚性质和酶活性进行了表征。使用 X 射线晶体学进行了原子分辨率的结构比较。虽然酶动力学显示无活性，但光谱探针并未在突变体中检测到任何明显的二级结构变化。突变体蛋白的 X 射线晶体学分析在 2 Å 分辨率下突出了一个水分子的重要性，该水分子在介导维持蛋白酶功能整体的分子间相互作用中发挥重要作用。总体而言，晶体学数据以及生物物理和酶学研究有助于解析 hHtrA2S276C 无活性的结构基础，这可能为进一步研究其与正常细胞功能异常的相关性，从而致病性奠定基础。
