Lin Yuan-Min, Liu Hsuan-Liang, Zhao Jian-Hua, Huang Chi-Hung, Fang Hsu-Wei, Ho Yih, Chen Wen-Yih
Department of Chemical Engineering and Biotechnology and Graduate Institute of Biotechnology, National Taipei University of Technology, 1 Sec. 3 ZhongXiao E. Rd., Taipei, Taiwan 10608.
Biotechnol Prog. 2007 May-Jun;23(3):577-84. doi: 10.1021/bp060380d. Epub 2007 Apr 17.
Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form a dimeric structure via a domain swapping process and then cause amyloid deposits in the brains of patients suffering from Alzheimer's disease. HCC monomer consists of a core with a five-stranded antiparallel beta-sheet (beta region) wrapped around a central helix. The connectivity of these secondary structures is: (N)-beta1-alpha-beta2-L1-beta3-AS-beta4-L2-beta5-(C). In this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500 K) and pH levels (2, 4, and 7) to gain insight into the domain swapping mechanism. The results show that high temperature (500 K) and low pH (pH 2) will trigger the domain swapping process of HCC. We further proposed that the domain swapping mechanism of HCC follows four steps: (1) the alpha-helix moves away from the beta region; (2) the contacts between beta2 and beta3-AS disappear; (3) the beta2-L1-beta3 hairpin unfolds following the so-called "zip-up" mechanism; and finally (4) the HCC dimer is formed. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of the alpha-helix from the beta-region, especially at low pH value. This is attributed to the fact that that low pH results in the protonation of the side chains of Asp, Glu, and His residues, which further disrupts the following four salt-bridge interactions stabilizing the alpha-beta interface of the native structure: Asp15-Arg53 (beta1-beta2), Glu21/20-Lys54 (helix-beta2), Asp40-Arg70 (helix-AS), and His43-Asp81 (beta2-AS).
人胱抑素C（HCC）是一种可形成淀粉样蛋白的蛋白质，已被证明通过结构域交换过程形成二聚体结构，进而在阿尔茨海默病患者大脑中导致淀粉样蛋白沉积。HCC单体由一个核心组成，该核心有一个五链反平行β折叠（β区域）环绕着一个中心螺旋。这些二级结构的连接性为：（N）-β1-α-β2-L1-β3-AS-β4-L2-β5-（C）。在本研究中，进行了各种分子动力学模拟，以研究单体HCC在不同温度（300和500K）和pH值（2、4和7）下的构象变化，从而深入了解结构域交换机制。结果表明，高温（500K）和低pH值（pH2）会触发HCC的结构域交换过程。我们进一步提出，HCC的结构域交换机制遵循四个步骤：（1）α螺旋从β区域移开；（2）β2和β3-AS之间的接触消失；（3）β2-L1-β3发夹按照所谓的“拉链式”机制展开；最后（4）形成HCC二聚体。我们的研究表明，高温可加速HCC的展开以及α螺旋从β区域的离开，尤其是在低pH值时。这归因于低pH导致天冬氨酸、谷氨酸和组氨酸残基侧链质子化，进而进一步破坏稳定天然结构α-β界面的以下四个盐桥相互作用：天冬氨酸15-精氨酸53（β链1-β链2）、谷氨酸21/20-赖氨酸54（螺旋-β链2）、天冬氨酸40-精氨酸70（螺旋-AS）和组氨酸43-天冬氨酸81（β链2-AS）。
