- 學位論文
葡萄糖轉運蛋白-1（GLUT1）中分子運輸途徑的預測研究
Prediction of molecular transport pathways in the human glucose transporter 1 (GLUT1)
摘要
醣類是身體重要的能源之一，但由於其水溶性的特性，需要透過細胞膜表面的特別蛋白通道，才能穿過脂質所構成的細胞膜來被細胞吸收利用。這個運輸通道對於葡萄糖具有獨特的專一性，只允許葡萄糖分子自由穿越細胞膜。葡萄糖運轉蛋白家族(GLUT family)在身體各組織細胞中細胞的表現數量不同，且運輸葡萄糖時的速度也不同，以此達到體內葡萄糖濃度的恆定。這些蛋白被稱為GLUT家族（GLUT family），依其被發現的順序被分別命名為GLUT1、GLUT2、GLUT3、GLUT4……依此類推。其中GLUT1主要分佈在紅血球，是目前被廣泛研究的細胞膜蛋白(membrane transporter)之一。但經由GLUT1運輸葡萄糖的分子機制目前仍有許多未被瞭解。 由於膜蛋白的結晶不易，至今仍未有實驗方法可以得到其高解析度的結構。因此以大腸桿菌的glycerol phosphate transporter為模本及glucose 6-phosphate translocase為中間模本，利用同源模擬的方法建立初始結構。再將此初始結構放到細胞膜的原子模型中，加入足量的水分子將其完整包覆，並調整成0.15M氯化鈉的生理環境，利用分子動力學模擬方法修飾此初始結構。根據化學交聯(chemical crosslinking)實驗結果，我們將G145及V328間的距離漸漸修飾調整到16 Å後，再移除此拉力並繼續模擬，使蛋白質可以回到其最喜歡且穩定的構形。利用umbrella sampling方法以及weighted histogram analysis method (WHAM)以計算得出平均力場(potential of mean force)，由結果可看出修飾過後的結構的自由能的確較低，G145 及 V328間的最佳距離是18 Å，且S148-V328, G145-L325 及 S148-L325在拉力移除後的模擬過程中，其距離也一直在6-16 Å間，與化學交聯實驗結果吻合。另外由嵌合模擬實驗結果可發現利用修飾完的結構能更準確的預測出葡萄糖結合位置。 利用一個新的嵌合(docking)模擬程序，可以找出一系列相鄰的配體(ligand)結合位置及其較佳的結合構形，如此可預測出葡萄糖運輸途徑(transport pathway)。另外，藉由得到沿著此運輸途徑受質與蛋白質間的結合自由能(binding free energy)可用來探討受質的選擇性。將修飾完的結構結合葡萄糖進行分子動力學模擬計算，以探討在葡萄糖運輸過程中GLUT1的構形變化。對於葡萄糖運輸蛋白的結構與其運輸過程有更清楚的瞭解可以幫助葡萄糖不平衡如乙型糖尿病的藥物開發研究。
並列摘要
Passive transport of glucose across the plasma membrane is mediated by members of the glucose transporter (GLUT/SLC2A) family which belongs to the major facilitator superfamily (MFS). GLUT1, known as the red blood cell glucose transporter, is one of the most extensively studied membrane transporters. However, the molecular mechanism of GLUT1-mediated glucose transport is still little clarified. The homology model of GLUT1(PDB code: 1SUK) was constructed using the crystal structure of E. coli glycerol phosphate transporter as the template and glucose 6-phosphate translocase as the intermediate. We have conducted molecular dynamics (MD) simulations of the GLUT1 in the full-hydrated POPC bilayer to refine the homology model based on chemical crosslinking data determined by di-cysteine mutagenesis. The distance between G145 and V328 was gradually restrained to 16 Å by umbrella sampling technique and then the restraint was removed, in this way, the GLUT1 was relaxed to its favorable conformation. The weighted histogram analysis method (WHAM) was employed for calculating the potential of mean force (PMF) profile, which indicated that the refined structure did have lower free energy. The minima of the PMFs located at the same point indicate that the optimal distance between G145 and V328 is around 18 Å. The distances of S148-V328, G145-L325 and S148-L325 after simulation are within 6-16 Å, which is consisted with the chemical crosslinking data. It was found that the predicted glucose binding sites generated from docking simulations on the refined structure were in better agreement with the existent experiments compared to those on the original homology structure. The glucose transport pathways were predicted by a novel docking scheme, SLITHER, which was modified from AutoDock 3.05. It can locate a series of juxtaposed ligand binding sites within such membrane channels, along with the most favorable conformation at each binding site. The binding free energies of substrates along the pathways can be used to investigate the mechanisms of substrate selectivity. The MD simulations of the GLUT1 with glucose in a full-hydrated POPC bilayer were conducted. The simulation results can be used to investigate the conformational changes upon glucose transport. Detailed knowledge of the structure of the glucose transporters will lead to advances in the understanding and therapeutics of glucose homeostasis disorders, including type 2 diabetes mellitus.