Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2
Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2
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DOI：
10.1096/fj.13-227256
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发表时间：
2013-08-01
期刊：
影响因子：
4.8
通讯作者：
Bose, Kakoli
中科院分区：
文献类型：
--
作者：
Chaganti, Lalith K.;Kuppili, Raja Reddy;Bose, Kakoli
HtrA 2是一种复杂的三聚体锥体线粒体丝氨酸蛋白酶，调节关键的生物学功能和疾病，包括细胞凋亡和癌症，是一个有前途的治疗靶点。它通过多种途径促进细胞凋亡，其复杂的机制仍然是难以捉摸的。现有的活化模型强调C-末端PDZ和蛋白酶结构域之间的相对分子内运动（PDZ-蛋白酶在非活性和静息状态下的塌陷），但未能明确地证明其作用的动力学。利用结构导向设计、分子生物学和蛋白质生物化学，我们获得了HtrA 2结构域和突变体的各种组合。构象变化和稳定性的特点，使用分子动力学模拟和光谱工具，而功能酶学划定其在调节酶催化的作用。定量福斯特共振能量转移显示，与其无活性单体对应物相比，三聚体HtrA 2中的分子内PDZ-蛋白酶距离较小（在37 ℃下分别约为21和22.3埃）。我们的研究结果强调了N-末端区域，寡聚化，和复杂的分子间PDZ-蛋白酶相互作用在适当的活性位点形成，酶-底物复合物稳定，因此HtrA 2功能的重要性。这些观察结果重新定义了现有的激活模型，并展示了一个独特的例子，精确的域间协调，可塑性和分子间接触如何导致不同的功能特性，从而提供了新的见解HtrA 2的结构，功能和动力学。
HtrA2, a complex trimeric pyramidal mitochondrial serine protease that regulates critical biological functions and diseases, including apoptosis and cancer, is a promising therapeutic target. It promotes apoptosis through multiple pathways, complex mechanisms of which are still elusive. The existing model of activation that emphasizes relative intramolecular movements between C-terminal PDZ and protease domains (PDZ-protease collapse in inactive and resting states) has not been able to unambiguously demonstrate dynamics of its actions. Using structure-guided design, molecular biology and protein biochemistry, we obtained various combinations of HtrA2 domains and mutants. Conformational changes and stability were characterized using molecular dynamics simulation and spectroscopic tools while functional enzymology delineated their roles in regulating enzyme catalysis. Quantitative Forster resonance energy transfer showed lesser intramolecular PDZ-protease distance in trimeric HtrA2 compared to its inactive monomeric counterpart (approximate to 21 and approximate to 22.3 angstrom, respectively, at 37 degrees C). Our findings highlight importance of N-terminal region, oligomerization, and intricate intermolecular PDZ-protease interaction in proper active-site formation, enzyme-substrate complex stabilization, and hence HtrA2 functions. These observations redefine the existing activation model and showcase a unique example of how precise interdomain coordination, plasticity, and intermolecular contacts lead to distinct functional properties and hence provide new insights into HtrA2 structure, function, and dynamics.