- 學位論文
以分子動力模擬探討動力蛋白之運作機制
Explore Dynein Mechanism by Molecular Dynamics Simulation
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摘要
馬達蛋白作為細胞中物質運輸的關鍵角色，參與了許多細胞活動如細胞分裂、胞器定位、複合蛋白移動等等。馬達蛋白分為三大類－肌球蛋白 (myosin) 、驅動蛋白 (kinesin) 及動力蛋白 (dynein)，其中不同於肌球蛋白與驅動蛋白單體的運動結構域 (motor domain) 僅由一個核苷酸結合位構成的，動力蛋白單體的運動結構域－AAA+環具有六個互相串聯的AAA+結構域 (ATPases associated with diverse cellular activities) ，代表可有多個核苷酸同時與蛋白結合，也因此衍伸了不同的行為機制。目前已知動力蛋白的機制由AAA1的核苷酸狀態控制，而要使運作循環得以開始的先決條件是AAA3中的ATP水解；過去的研究指出，在具有完整功能的AAA1、AAA3與AAA4中，AAA1與AAA3這兩個位點能否水解ATP會直接影響動力蛋白的移動能力，而AAA4中發生水解與否則影響不大，因此本研究將目光聚焦在AAA1與AAA3，首先觀察水解產物ADP的結合位，接著利用增強採樣模擬了解ADP的釋放過程與路徑，使現有的機制更加完善。 平衡的結構顯示兩位點會由相同的催化基序 (motif) 構成ADP結合位，且結合位與ADP間形成的氫鍵數量並無明顯差異，但在AAA1中與ADP形成氫鍵的殘基中有四個位在負責結合功能的Walker A基序，而AAA3中只會有兩個。關於ADP的釋放行為，兩組增強採樣模擬的結果皆顯示AAA1擁有更高的勢能壘， ADP會較難從AAA1脫離；至於ADP離開所走的路徑，氫鍵分析的結果說明，除了構成結合位的殘基之外，都會有一個在AAAS－H7上的殘基參與釋放過程，代表兩位點擁有相同ADP釋放路徑，不過此過程中ADP會以不同的方式斷開與結合位間的氫鍵，一種是許多氫鍵一起被斷開，另一種則是逐一斷開，這可能是最初結合位構成差異所導致的，然而確切原因還需深入探討才可得知。總而言之，這項研究確定了ADP在AAA1及ＡAA3中的結合位構成，計算出了ADP離開AAA1與AAA3兩位點所需的能量以及釋放的路徑，並發現在類似的條件下 AAA1比起AAA3擁有更高的勢能壘需要克服。
並列摘要
Motor proteins play a critical role in intracellular material transport, participating in numerous cellular activities such as cell division, organelle positioning, and the movement of protein complexes. Motor proteins are classified into three major categories: myosin, kinesin, and dynein. Unlike the motor domains of myosin and kinesin monomers, which consist of only one nucleotide-binding site, the motor domain of dynein monomers—the AAA+ ring—features six tandemly linked AAA+ domains (ATPases associated with diverse cellular activities), allowing multiple nucleotides to bind to the protein simultaneously. This leads to diverse behavioral mechanisms. Currently, it is known that dynein's mechanism is controlled by the nucleotide state of AAA1, with the prerequisite for initiating the operational cycle being ATP hydrolysis in AAA3. Previous research has indicated that in the fully functional AAA1, AAA3, and AAA4 sites, the ability to hydrolyze ATP of AAA1 and AAA3 directly affects dynein's motility, while hydrolysis in AAA4 has a minimal impact. Therefore, this study focuses on AAA1 and AAA3, first observing the ADP binding sites and then using enhanced sampling simulations to understand the ADP release process and pathways, thereby refining the existing mechanism. Equilibrium structures show that both sites form ADP binding sites with the same catalytic motif and the number of hydrogen bonds between the binding site and ADP does not differ significantly. However, in AAA1, four residues forming hydrogen bonds with ADP are located in the Walker A motif which is responsible for binding functions, while in AAA3, only two residues are involved. Regarding ADP release behavior, results from two sets of enhanced sampling simulations show that AAA1 has a higher energy barrier, making ADP more difficult to dissociate from AAA1. As for the pathway of ADP release, hydrogen bond analysis indicates that, besides the residues forming the binding site, a residue on AAAS-H7 participates in the release process, indicating that both sites have the same ADP release pathway. However, during this process, ADP breaks hydrogen bonds with the binding site in different ways: in one, many hydrogen bonds break simultaneously, while in the other, they break one by one. This may be due to the initial differences in binding site composition, but the exact reasons require further investigation. In summary, this study identifies the composition of ADP binding sites in AAA1 and AAA3, calculates the energy required for ADP to leave the two sites and their release pathways, and found that under similar conditions, AAA1 has a higher energy barrier to overcome compared to AAA3.
參考文獻
1. Kull, F.J. and S.A. Endow, Force generation by kinesin and myosin cytoskeletal motor proteins. Journal of Cell Science, 2013. 126(1): p. 9-19.
2. Titus, M.A., Myosin-driven intracellular transport. Cold Spring Harbor perspectives in biology, 2018. 10(3): p. a021972.
3. DePina, A.S. and G.M. Langford, Vesicle transport: the role of actin filaments and myosin motors. Microscopy research and technique, 1999. 47(2): p. 93-106.
4. Miki, H., Y. Okada, and N. Hirokawa, Analysis of the kinesin superfamily: insights into structure and function. Trends in cell biology, 2005. 15(9): p. 467-476.
5. Hirokawa, N. and R. Takemura, Kinesin superfamily proteins and their various functions and dynamics. Experimental cell research, 2004. 301(1): p. 50-59.