阿拉伯芥磷酸鹽轉運蛋白AtPHT1;1關鍵氨基酸殘基的結構功能分析

Structure-Function Analysis Reveals Amino Acid Residues of Arabidopsis Phosphate Transporter AtPHT1;1 Crucial for Its Activity

廖雅韻(Ya-Yun Liao)

指導教授 ： 潘榮隆 ； 邱子珍

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2019年)

摘要

磷為植物生長與發育中之必要大量元素，在植物體中，磷的獲取主要仰賴植物根中磷酸鹽轉運蛋白運輸無機磷酸鹽。為了瞭解氫離子偶聯磷酸鹽共同轉運蛋白之運輸機制，本研究應用結構-功能分析，研究阿拉伯芥磷酸鹽轉運蛋白1; 1（AtPHT1; 1）在植物根中磷的獲取機制。首先，利用已解構之印度梨型孢真菌磷酸鹽轉運蛋白（PiPT）的3D晶體結構為模板，預測阿拉伯芥磷酸鹽轉運蛋白1; 1的二級和三級結構，挑選出可能參與阿拉伯芥磷酸鹽轉運蛋白1; 1活性之28個氨基酸殘基，其次，將28個氨基酸殘基突變成丙氨酸，分別表現於高親和力磷酸鹽轉運蛋白缺陷之酵母菌pam2突變體和阿拉伯芥pht1; 1突變體，分析各氨基酸之互補能力效應。最後，整合結構和功能性互補分析，並提出阿拉伯芥磷酸鹽轉運蛋白1; 1運輸氫離子與磷酸鹽之機制，其中，D35、D38、R134和D144氨基酸參與跨膜氫離子運輸，而Y312和N421氨基酸參與初始磷酸鹽運輸，當磷酸鹽進入結合位置時，兩個芳族氨基酸Y145和F169與結合位置中Q172、W304、Y312、D308和K449氨基酸產生分子內氫鍵，維持了磷酸鹽結合位置之結構穩定，隨後磷酸鹽與正電K449氨基酸相互作用，促使磷酸鹽從結合位置釋出，此外，D38、D93、R134、D144、D212、R216、R233、D367、K373和E504氨基酸，可以形成內部靜電相互作用來穩定阿拉伯芥磷酸鹽轉運蛋白1; 1之結構整體和適應性，綜合結構和功能性互補分析，這個研究提供了一個運輸氫離子與磷酸鹽機制的模型，闡明植物磷酸鹽轉運蛋白的運輸機制。

關鍵字

磷酸鹽轉運蛋白 ； 阿拉伯芥 ； 主要促進家族 ； 氨基酸殘基 ； 結構

並列摘要

Phosphorus (P), an essential plant macronutrient, is acquired in the form of inorganic phosphate (Pi) by transporters located at the plasma membrane of root cells. To decipher the Pi transport mechanism, Arabidopsis thaliana Pi transporter 1;1 (AtPHT1;1), the most predominantly H+-coupled Pi co-transporter in the root, was selected for structure-function analysis. We first predicted its secondary and tertiary structures on the basis of the Piriformospora indica Pi transporter (PiPT) and identified 28 amino acid residues potentially engaged in the activity of AtPHT1;1. We then mutagenized these residues into alanine and expressed them in the yeast pam2 mutant defective in high-affinity Pi transporters and Arabidopsis pht1;1 mutant, respectively, for functional complementation validation. We further incorporated the functional characterization and structure analyses to propose a mechanistic model for the function of AtPHT1;1. We showed that D35, D38, R134 and D144, implicated in H+ transfer across the membrane, and Y312 and N421, involved in initial interaction and translocation of Pi, are all essential for its transport activity. When Pi enters the binding pocket, the two aromatic moieties of Y145 and F169 and the hydrogen bonds generated from Q172, W304, Y312, D308, and K449 can build a scaffold to stabilize the structure. Subsequent interaction between Pi and the positive residue of K449 facilitates its release. Furthermore, D38, D93, R134, D144, D212, R216, R233, D367, K373, and E504 may form internal electrostatic interactions for structure ensemble and adaptability. This study offers a comprehensive model for elucidating the transport mechanism of a plant Pi transporter.

並列關鍵字

Phosphate Transporter ； AtPHT1 ； 1 ； Arabidopsis thaliana ； Major Facilitator Superfamily ； Amino Acid Residues ； Structure

參考文獻

Ai, P., Sun, S., Zhao, J., Fan, X., Xin, W., Guo, Q., Yu, L., Shen, Q., Wu, P., Miller, A. J., and Xu, G. (2009) Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. Plant J., 57: 798-809

Ayadi, A., David, P., Arrighi, J. F., Chiarenza, S., Thibaud, M. C., Nussaume, L., and Marin, E. (2015) Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. Plant Physiol., 167: 1511-1526

Baek, D., Chun, H. J., Yun, D. J., and Kim, M. C. (2017) Cross-talk between phosphate starvation and other environmental stress signaling pathways in plants. Mol. Cells, 40: 697-705

Bayle, V., Arrighi, J. F., Creff, A., Nespoulous, C., Vialaret, J., Rossignol, M., Gonzalez, E., Paz-Ares, J., and Nussaume, L. (2011) Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. Plant Cell, 23: 1523-1535

Bun-Ya, M., Nishimura, M., Harashima, S., and Oshima, Y. (1991) The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol. Cell Biol., 11: 3229-3238