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

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研究生：	廖雅韻 Liao, Ya-Yun
論文名稱：	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
指導教授：	潘榮隆 Pan, Rong-Long 邱子珍 Chiou, Tzyy-Jen
口試委員:	劉姿吟 Liu, Tzu-Yin 黃蘊慈 Huang, Yun-Tzu 林士鳴 Lin, Shih-Ming
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2019
畢業學年度：	108
語文別：	英文
論文頁數：	53
中文關鍵詞：	磷酸鹽轉運蛋白、阿拉伯芥、主要促進家族、氨基酸殘基、結構
外文關鍵詞：	AtPHT1;1, Major Facilitator Superfamily, Amino Acid Residues
相關次數：	點閱：3 下載：0
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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。

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.

Table of Contents
Introduction.......................................................1
Materials and Methods..............................................7
Plant materials and growth conditions..............................7
Plant materials and growth conditions..............................7
Arabidopsis transformation.........................................7
Measurement of phosphate contents..................................8
Agrobacterium-mediated infiltration of tobacco leaves..............8
Yeast manipulation, growth complementation and Pi transport assay..9
Isolation of membrane proteins from yeast..........................9
Total protein extraction from Arabidopsis.........................10
SDS-PAGE and immunoblot analysis..................................10
Bioinformatics analysis of AtPHT1;1...............................11
Results...........................................................12
Structure prediction of AtPHT1;1..................................12
Selection of potential key amino acid residues involved in the Pi transport activity of AtPHT1;1....................................13
Functional analysis of AtPHT1;1 in yeast pam2 mutant..............13
Functional analysis of AtPHT1;1 in Arabidopsis mutant.............15
TM-localized amino acid residues of AtPHT1;1 crucial for Pi transport.........................................................16
Key amino acid residues of AtPHT1;1 on the extracellular side.....17
Key amino acid residues of AtPHT1;1 on the cytoplasmic side.......18
Discussion........................................................20
Essential residues within TM domain...............................21
1.    Essential residues for Pi transport.......................21
2.    Essential residues for H+ transfer........................22
Essential residues at bilayer interfaces on extracellular or cytoplasmic sides.................................................23
1.    Essential residues for conformational changes, internal electrostatic interactions, and structural stability..............23
2.    Residues for other functionalities........................24
A mechanistic model of Pi transport of AtPHT1;1...................25
Conclusions.......................................................26
Future perspectives...............................................27
References........................................................29
Figures...........................................................34
Figure 1. 3D structure of AtPHT1;1................................35
Figure 2. Topology of AtPHT1;1....................................36
Figure 3. Characterization of AtPHT1;1 expression and function in S. cerevisiae........................................................37
Figure 4. Evaluation of TM-localized amino acid residues of AtPHT1;1 by functional analyses in yeast and Arabidopsis...................38
Figure 5. Evaluation of amino acid residues on the extracellular side of AtPHT1;1 by functional analyses in yeast and Arabidopsis.......39
Figure 6. Evaluation of amino acid residues on the cytoplasmic side of AtPHT1;1 by functional analyses in yeast and Arabidopsis..........40
Figure 7. Proposed mechanism of Pi transport of AtPHT1;1..........41
Appendix..........................................................42
Table S1. List of primer sequences................................43
Table S2. The corresponding amino acid residues in AtPHT1;1, ScPHO84, and PiPT..........................................................44
Table S3. Homologous residues of A-motif in YajR, XylE, PiPT, and AtPHT1;1..........................................................45
Table S4. The proposed functions of amino acid residues in MFS....46
Figure S1. Sequence alignment of the AtPHT1;1 homologues..........47
Figure S2. Putative hydrogen bonds in the inward facing occluded state of AtPHT1;1.......................................................48
Figure S3. Prediction of intrinsically disordered regions in AtPHT1;1..........................................................49
Figure S4. Complementation analysis of pht1;1 mutants.............50
Figure S5. Immunoblot analysis of AtPHT1;1 variants expressed in yeast pam2..............................................................51
Figure S6. Immunoblot analysis of AtPHT1;1 variants expressed in Arabidopsis   pht1;1 mutants......................................52
Figure S7. Plasma membrane localization of YFP-tagged AtPHT1;1 variants expressed in tobacco leaves..............................53

                                

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