阿拉伯芥(Arabidopsis thaliana) PHT1;1是一種高親和力的磷酸轉運蛋白，屬於主要的易化因子超家族(major facilitator superfamily; MFS)。主要的易化因子超家族則是繼發性主動運輸蛋白家族(secondary active transporters)中最大的一類。同向轉運蛋白PHT1;1會將質子和無機磷酸鹽以同樣的方向從土壤中轉運到植物體內。因為磷酸對植物的生長非常重要，理解PHT1;1轉運磷酸的機制能讓我們有機會提高植物對磷酸的轉運速率，進而幫助植物的生長。但是到目前為止，PHT1;1的磷酸轉運機制還不是很清楚，PHT1;1的晶體結構也尚未解出。我們發現PiPT——印度梨型孢真菌(Piriformospora indica)磷酸轉運膜蛋白，和PHT1;1有高達38%的序列同一性(sequence identity)，於是我們利用PiPT作為模版建立了PHT1;1的同源模擬結構 (homology modeling)，並且我們用這個結構進行了一系列分子動力學模擬(molecular dynamics simulations; MD simulations)來研究PHT1;1的磷酸轉運機制。在這些模擬過程中，我們特別考慮了細胞膜兩邊質子濃度的差異，並且根據細胞膜兩邊不同的pH值來決定PHT1;1上帶電胺基酸的質子化狀態(protonation state)。我們發現Asp38和Asp308的質子化狀態對磷酸的釋放非常重要。在磷酸轉運過程中，兩個質子可以分別通過兩個水通道和H2PO4- 一同被轉運。這兩個質子會分別將Asp38 和 Asp308 質子化，稍後再釋放到水中。此處質子化及去質子化均與磷酸結合，釋放及其所造成的構型變化相關。去質子化的Asp308會讓H2PO4- 離開原本的結合位點，使得PHT1;1產生形變。H2PO4- 離開Asp308附近之後，會逐步和Gln85、His441、Asp144、 Ser153、Thr150 發生作用，被釋放到細胞內。在磷酸釋放的過程中，PHT1;1的結構變化可以透過適當的投影方法在低維度的空間中觀測到。這個低維度的空間是以朝細胞膜外打開(outward facing)的PHT1;1結構最主要的兩個或三個運動模式(normal modes)為軸來建構成的。而這些運動模式是由各向異性網路模型(anisotropic network model; ANM)計算得到。這種結構的變化揭示了磷酸轉運過程中的機械化學協同作用(mechanochemical coupling)：磷酸與蛋白質的結合以及結構的變化導致了重要氨基酸質子化狀態改變之間的協同關係。除此之外，我們也發現在磷酸被釋放出PHT1;1的過程中，在Glu154 and Lys99 之間新形成的鹽橋可能會誘發PHT1;1形成朝細胞膜外打開的結構狀態。

The Arabidopsis thaliana PHT1;1 is a high-affinity phosphate transporter and belongs to the major facilitator superfamily (MFS), one of the largest groups of secondary active transporters. PHT1;1 as a symporter transports both phosphate and protons and plays an important role in phosphates uptake from soil and is thus vital for plant growth. Understanding the mechanism of phosphate transport by PHT1;1 paves a way for us to improve the efficiency of phosphate uptake in plants. However, the mechanism of PHT1;1 transport is currently still unknown. On the basis of the crystal structure of P. indica phosphate transporter (PiPT) that shares a similar sequence with PHT1;1 (38% sequence identity), we built the homology structural model of PHT1;1 and performed molecular dynamics (MD) simulations to study the transport mechanism. By carefully considering protonation states of ionizable residues according to proton gradient across the membrane in our MD simulations, we suggest that the protonation states of Asp38 and Asp308 play important roles in releasing phosphate. Through two water channels, two protons are transferred along with H2PO4- discharge-induced conformational changes and deprotonation of Asp38/Asp308. After leaving Asp308, the phosphate is escorted by Gln85, His441, Asp144, Ser153, and Thr150 toward its release into the cytosol. The conformational changes (states) during the phosphate release can be displayed in a plane or space spanned by the slowest 2 or 3 normal modes, revealing a mechanochemical coupling between phosphate binding/exit and conformational change-triggered (de)protonation of key residues. The formation of outward-facing-like structure could be triggered by a newly formed salt bridge between Glu154 and Lys99 during the phosphate release.

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