以分子動態模擬探討人類半胱胺酸蛋白酶抑制蛋白功能區域交換的機制

目前已發現人類體內約有20多種蛋白質單體會聚集，進而形成類澱粉纖維。人類半胱胺酸蛋白酶抑制蛋白 (human cystatin C，HCC)為上述20多種蛋白質的其中之一，並且已被證實會經由功能區域交換(domain swapping)的機制形成雙體，進而在患有阿茲海默症病人的腦部形成類澱粉沉積。HCC的結構是由5個逆向平行的β摺板所組成的β區域(β-region)圍繞著1個垂直的α螺旋所構成，其二級結構由N端至C端依序為(N)-β1-α-β2-L1-β3-AS-β4-L2-β5-(C)（其中L1、AS、及L2均為連結這些二級結構的迴圈）。HCC雙體結構主要是由二個單體互相交換β1-α-β2的區域結構所形成的。先前的研究明確指出，提高溫度與降低pH值會引發HCC功能區域交換。因此，本研究利用分子動態模擬研究HCC單體於不同溫度(300及500K)及不同pH值(2、4、及7)下的結構變化，進而洞察其功能區域交換的機制。研究結果發現，於高溫及低pH值(500K及pH2)的條件下容易發生功能區域交換，並且從中可推論HCC功能區域交換的步驟為：（1）α先脫離β區域、（2）β2與β3及AS之間的接觸消失、（3）接著造成β2-L1-β3這段β髮夾依照”zip-up”的機制瓦解、及（4）最後形成HCC雙體。研究結果亦顯示，在β2與β3分離之前，α得先脫離β區域，這意味著此一步驟為HCC 形成功能區域交換之前的關鍵步驟。由我們的結果中發現，升高溫度會加速α脫離β區域，特別是在低pH值的環境下。這可歸因於低pH值造成Asp、Glu、及His三個胺基酸側鏈基團的質子化，進而破壞下列四對原本維持α與β區域間的鹽橋作用力：Asp15-Arg53 (β1-β2)、Glu21/20-Lys54 (α-β2)、Asp40-Arg70 (α-AS)、與His43-Asp81 (β2-AS)。

關鍵字

單體；聚集；類澱粉纖維；人類半胱胺酸蛋白酶抑制蛋白；功能區域交換；雙體；阿茲海默症； zip-up ；髮夾；鹽橋；質子化

並列摘要

More than 20 human proteins are known to form aggregations and amyloid fibrils in vivo. Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form dimeric structure via domain swapping and further cause amyloid deposits in the brains of patients suffering from Alzheimer’s disease. HCC monomer consists of a core with a five-stranded anti-parallel β-sheet (β-region) wrapped around a central helix. The connectivity of these secondary structures from N to C-terminus is: (N)-β1-α-β2-L1-β3-AS-β4-L2-β5-(C), where L1, AS, and L2 are loops connecting these secondary structures. The dimeric HCC is formed by exchanging the β1-α-β2 local structures from two monomers. Previous studies have shown that high temperature and low pH conditions may trigger domain swapping of HCC. Therefore, in this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500K) and pHs (2, 4, and 7) and to gain insight into the domain swapping mechanism. The results show that high temperature (500K) and low pH (pH2) will trigger domain swapping of HCC. We further proposed that the domain swapping mechanism of HCC is as follows: (1) the α-helix moves away from β-region; (2) the contacts between β2 and β3-AS disappear; (3) the β2-L1-β3 hairpin unfolds following the so-called “zip-up” mechanism; and (4) the HCC dimer is formed. Our results also indicate that the moving of α-helix away from β-region is the initiate step for domain swapping of HCC. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of α-helix from β-region, especially at low pH value. It is attributed to that low pH condition 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 α-β interface of the native structure: Asp15-Arg53 (β1-β2), Glu21/20-Lys54 (α-β2), Asp40-Arg70 (α-AS), and His43-Asp81 (β2-AS).

並列關鍵字

monomer ； aggregation ； amyloid fibril ； human cystatin C ； domain swapping ； dimeric ； Alzheimer’s disease ； zip-up ； β-hairpin ； salt bridge ； protonatio

參考文獻

Chen CW, Lin JC, Liu HL. Molecular dynamics simulations of the tetramerization domain of Shaker and Kv1.1 potassium channels. J. Chin. Inst. Chem. Eng. 2005, 36. 649-660.

Liu HL, Hsieh WC. Molecular dynamics simulations to investigate the thermal unfolding behaviors of N-carbamyl-D-amino acid amidohydrolase. J. Chin. Inst. Chem. Eng. 2005, 36. 185-194.

Abrahamson M. Molecular basis for amyloidosis related to hereditary brain hemorrhage. Scand. J. Clin. Lab. Invest. Suppl. 1996, 226. 47-56.

Abrahamson M, Dalboge H, Olafsson I, Carlsen S, Grubb A. Efficient production of native, biologically active human cystatin C by Escherichia coli. FEBS Lett. 1988, 236. 14-18.

Abrahamson M, Grubb A. Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy. Proc. Natl. Acad. Sci. U S A. 1994, 91. 1416-1420.

國際替代計量

以分子動態模擬探討人類半胱胺酸蛋白酶抑制蛋白功能區域交換的機制

未授權

主題瀏覽