Dynamics, Conformational Transition and Drug Interactions of P-glycoprotein upon ATP Binding

由於ATP-dependent efflux pumps過度表現引起多重抗藥性(multidrug resistance, or MDR)，造成藥物在體內累積減少，在臨床上，尤其影響了化學治療的療效，其中最具代表性的即是P-glycoprotein（Pgp），其為人體MDR1基因的產物，屬於ATP-binding cassette (ABC) family。為改善抗藥性在治療上帶來的問題，因此設計P-glycoprotein的抑制劑便成為重要的課題。運用蛋白質結構從事新藥開發，已是目前受到廣泛運用的一種有效途徑。 由於Pgp屬於細胞膜蛋白，而細胞膜蛋白的表現不易，且結晶條件不易尋找，因此至今仍未能有實驗方法獲得高解析度結構。然而，大腸桿菌中的lipid A transporter – MsbA，為Pgp的homologue，序列比對達30~31%的相似性，最近已由X-ray方法解出結構，提供了以同源模擬方法建立Pgp結構的適當模本。同源模擬法建立的Pgp初始結構有三部分的缺漏，包括連接區段（linker region, residue 627-693）、部分的N端和C端序列（residue 1-33, 1272-1280），因此我們運用生物資訊的方法，將Pgp建立完整序列，並以分子動力學模擬方法修飾Pgp結構，再將此結構置於細胞膜的原子模型中，並加入足量的水分子將Pgp充分包覆，將系統設定為0.15M氯化鈉的生理相容環境，分別進行Pgp在結合ATP和未結合ATP的條件下的達9 ns的分子動力學計算。 分子動力學的模擬方法助益於了解微觀下Pgp的結構資訊，在我們的分子模擬過程中，Pgp不僅在細胞膜環境下穩定存在，並且藉由此平衡系統進一步的研究ATP的鍵結對Pgp的結構變化與動力研究，模擬結果顯示Pgp在ATP結合的條件下，確實對Pgp的穿膜結構域（transmenbrane domains）有不同於未結合ATP下的結構變化，再經由進一步的結構分析，發現此結構變化縮小了穿膜結構域的中央通道孔徑，此分析結果可能相關於受質的釋出。除此之外，再運用嵌合模擬（docking simulation）方法探討Pgp在ATP結合後的動態變化對受質以及抑制劑所造成的影響，並預測藥物分子與Pgp之間相關結合位置。修飾後的Pgp結構除了對抗藥性機轉的解析有進一步的了解外，相信對於日後的藥物設計亦有所幫助。The overexpression of P-glycoprotein (Pgp) is one of the major causes of multidrug resistance (MDR) in cancer chemotherapies. Until now, it remains very challenging to obtain the high resolution Pgp structure and currently only low resolution electron microscopy structure is available. The recent determination of the X-ray crystallographic structure of a batcterial lipid A transporter, MsbA, has provided good structure templates for homology modeling of P-gp, and in our lab this model is used to further construct a full-length Pgp structure. We have conducted molecular dynamics (MD) simulation of the full-length Pgp in an excessively hydrated POPC bilayer. The starting structure was from a homology model but the N-terminal, C-terminal and the linker region were missing and therefore we have mended their structure. This mended structure was then energy minimized. The full-length Pgp was embed in the POPC bilayer and water was then added along the membrane normal direction to fully soak the whole system. The whole system was simulated in an environment closer to physiological condition with the salt concentration of 0.15M. Both free and ATP-bound Pgp forms of Pgp were simulated. The total simulation time was 9 ns for both simulations. On the other hand, we have conducted the docking simulation to find out the major transmembrane helices for drug binding. MD simulation provides very detailed structural information at atomic resolution. It was shown from our simulations that the overall architecture of the P-glycoprotein is stable in a realistic lipid bilayer environment, and the simulation results have allowed us to investigate the conformational changes of Pgp upon ATP binding in the efflux process. Not only the binding of ATP indeed drives the conformational changes in the TMDs, but also the helix rearrangements reduce the space of TMD center pore to mediate drug transportation. The mechanistic detail of the transport cycle upon binding ATP will be helpful to interpret remote connection between the binding of ATP and the subsequent release of substrates. The refined structure models of Pgp by our MD simulations may be used as the basis for designing Pgp inhibitors or “Pgp-ignoring drugs”.目錄 i 中文摘要 ii 英文摘要 iii 第一章 緒論 1 第二章 研究目的 3 第三章 P-glycoprotein的抗藥性機轉模式 4 3.1 Alternating Catalytic Sites Model 6 3.2 ATP Switch Model 8 第四章 P-glycoprotein的結構生物學 16 4.1 電子顯微鏡（electron microscopy，EM） 17 4.2 Homology Model 18 第五章 P-glycoprotein完整序列的結構建立 21 5.1 同源模擬結構缺漏部分之建構 21 5.2 Pgp完整結構之最佳化 22 第六章 P-glycoprotein未結合藥物的分子動力學研究 25 6.1 建構P-glycoprotein於POPC細胞膜環境 25 6.2 進行系統的能量最小化 (Energy Minimization) 26 6.3 分析P-glycoprotein的運動與系統平衡過程 26 第七章P-glycoprotein結合與未結合ATP下之結構變化與動力研究 35 7.1 P-glycoprotein標定分子模擬方法 35 7.2 P-glycoprotein結合鎂離子與ATP 37 7.3 探討ATP對P-glycoprotein的影響 38 第八章P-glycoprotein結構動態變化對藥物分子結合影響 72 8.1 選擇P-glycoprotein的受質與抑制劑 72 8.2 分析P-glycoprotein的嵌合模擬結果 75 第九章 結論 108 第十章 參考文獻 11