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研究生： 吳孟純
Wu, Meng-Chun
論文名稱： 綠膿桿菌中訊息感知激酶之磷酸接收區晶體結構推測與磷酸轉移酶HptB進行訊息傳遞之結合構型
Crystal structure of the receiver domain of sensor histidine kinase from Pseudomonas aeruginosa suggest a binding model with phosphotransfer HptB for signal transduction
指導教授： 陳俊榮
Chen, Chun-Jung
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生物科技研究所
Institute of Biotechnology
論文出版年： 2014
畢業學年度： 102
語文別： 英文
論文頁數： 54
中文關鍵詞： 雙元件系統 、PA1611磷酸接收區 、磷酸接收區與磷酸轉移酶HptB複合體模型
外文關鍵詞： Two-component system, PA1611 receiver domain, model of receiver domain and HptB complex
相關次數： 點閱：100  下載：1 
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    • 雙元件系統是細菌體內的訊息傳遞系統，可使細菌感受外界環境刺激並對此進行反應調控。在銅綠假單孢菌中，由磷酸轉移酶HptB所參與的訊息傳遞路徑被發現與調控菌體的泳動能力有關。此訊息傳遞路徑主要由訊息感知激酶PA1611、磷酸轉移酶HptB及反應調節酶PA3346組成，扮演訊息的磷酸根主要由PA1611中的HATPase 催化ATP而來，並結合至HisKA區位，進而由PA1611磷酸接收區位REC上的保留性Asp胺基酸接收。在進行訊息傳遞時，磷酸接收區上的磷酸根會被傳遞到磷酸轉移酶HptB上，進一步由反應調節酶PA3346接收後調控相關基因表現。
    為了瞭解磷酸接收區與磷酸轉移酶之間訊息傳遞的機制，我們成功以同步輻射X光繞射解析得到1.5 Å的高解析繞射數據並進而解構出磷酸接受區的蛋白質晶體結構。經過分析後，我們發現此蛋白質的晶體結構呈現典型的磷酸接收區所具有(β/α)5 摺疊構型，並且帶有鎂離子結合於活化位中。此鎂離子主要是跟兩個具保留性的胺基酸Asp16、Asp59及Arg61進行鍵結，而此二價離子的結合似乎可調節活化位胺基酸Asp59的支鏈擺位，使此胺基酸得以與磷酸根受體結合。藉由電腦模擬的方式，我們得到了此磷酸接收區與磷酸轉移酶在進行訊息傳遞時可能形成的複合體結構，藉此模型我們發現兩個蛋白之間的結合面主要是由磷酸轉移酶的B、C、D螺旋與磷酸接收區的1號螺旋及連接β折疊與α-螺旋的環圈所形成，而此兩個蛋白在活化位上的疏水性作用力與表面電位互補或許是促進兩者形成複合體的作用力。

    Two-component system (TCS) is the signal transduction system for bacteria to sense and response to their environment. In Pseudomonas aeruginosa (P. aeruginosa), the histidine-containing phosphotansferase B (HptB) -mediated signal transduction pathway is found to regulate the swarming ability of the bacterium. The TCS of P. aeruginosa consists of a hybrid sensor histidine kinase (HK) PA1611, histidine-containing phosphotransferase B (HptB) and a response regulator (RR) PA3346. The phosphoryl group from ATP catalysis by the histidine kinase-like ATPase domain (HATPase) of PA1611 and binds to the histidine kinase phosphoacceptor domain (HisKA), and is subsequently received by the conserved Asp residue of the receiver domain (REC) of PA1611. In signal transduction, the phosphoryl group on the REC would be transferred to HptB, and further received by PA3346 to regulate the gene expression.
    To reveal the mechanism of signal transduction between REC and HptB, we have determined the crystal structure of REC at high resolution 1.5 Å with synchrotron X-ray. The overall structure of REC domain shows the typical (β/α)5 topology with the Mg2+ bound inside the active site. The bound Mg2+ is interacting with two conserved residues Asp16 and Asp59, as well as Arg61. This binding of divalent ion may mediate the side-chain position of the active residue Asp59 for receiving the phosphoryl group of the REC domain. The suggested binding model of REC and HptB calculated with the HADDOCK docking program shows that the binding interface is formed by helices B, C and D with positive charges of HptB, and the helix α1 and loops with negative charges connecting β1-α1, β2-α2, β3-α3 and β4-α4 of the REC domain. The electrostatic binding surfaces of HptB with positive charges and REC with negative charges compliment with the surrounding hydrophobic regions and may contribute to complex formation and stability.

    Table of Contents Chapter 1. Introduction 1 1.1 Two-Component Regulatory Systems (TCS) 1 1.2 Sensor Histidine Kinase (HK) 1 1.3 Histidine-containing Phosphotransferase (Hpt) 3 1.4 Response Regulator (RR) 3 1.5 TCS in Pseudomonas aeruginosa 4 1.6 The Composition of PA1611 5 1.7 Purpose of the Study 5 Chapter 2. Material and Methods 6 2.1 Cloning of REC Domain 6 2.2. Small-Scale Expression Test 6 2.3 Large-Scale Expression 7 2.4 Protein Purification 8 2.5 Gel-Filtration Chromatography (GFC) 8 2.6 Synchrotron Radiation Circular Dichroism (SRCD) 9 2.7 Dynamic Light Scattering (DLS) Measurement 11 2.8 Protein Crystallization 11 2.9 Data Collection and Structure Determination 12 2.10 The HADDOCK Docking Protocol 13 Chapter 3. Results 15 3.1 Protein Purification and Structural Characterization 15 3.2 The Secondary Structure of REC Domain 16 3.3 Crystallographic Data and Structure Determination 16 3.4 REC Structure Overview 17 3.5 The Docking Model of REC and HptB Complex 18 Chapter 4. Discussion 20 4.1 Comparison of Other REC Structures 20 4.2 The Divalent Ion Binding in Receiver Domain 21 4.3 The Suggested Binding Model of the Receiver Domain and Phosphotransfer HptB 22 Reference 26 List of Tables Table. 1 The DNA primers used in the study. 30 Table. 2 The recipe of 15 % SDS-PAGE. 30 Table. 3 X-ray data collection statistics of PA1611 receiver domain (REC). 31 Table. 4 The top 5 clusters result of REC and HptB docking complex from HADDOCK. 32 List of Figures Fig. 1 Three types of Two-component regulatory system. 33 Fig. 2 The HptB-mediated signal transduction pathway in P.aeruginosa. 34 Fig. 3 The Sephacryl-200 (S200) calibration curve 35 Fig. 4 The SRCD result of REC domain. 36 Fig. 5 The measurement of suitable conditions for protein crystallization by DLS. 37 Fig. 6 The crystals of PA1611 REC domain. 38 Fig. 7 Small-scale expression and purification trial. 39 Fig. 8 Large-scale protein purification. 40 Fig. 9 Sephacryl-200 purification result. 41 Fig. 10 The DLS measurement of REC domain in dH2O and 20 mM Tris, pH 8.0 42 Fig. 11 The electron density map of Mg2+-binding site. 43 Fig. 12 Overview of the structure of REC domain. 44 Fig. 13 The Mg2+- binding site of REC 45 Fig. 14 The docking model of PA1611REC and HptB complex. 46 Fig. 15 Sequence alignment of Mg2+- containing receiver domains. 47 Fig. 16 The comparison of Mg2+-receiver domains 48 Fig. 17 The divalent ion play an important role in activate the conserved phosphorylated Asp residue to move inward to the active site. 49 Fig. 18 The electrostatic potential surface of HptB and REC. 50 Fig. 19 The comparison of PA1611REC/HptB and SLN1/YPD1 complex. 51 Fig. 20 The distance of active site between Hpt and receiver domain. 51 Fig. 21 The hydrophobic surrounding of the active site in HptB and PA1611REC. 52 Fig. 22 The electrostatic potential surface of PA1611 REC and HptB complex. 52 Fig. 23 The differences of active sites in complex form of HptB/ PA1611REC and individual HptB and PA1611REC. 53 Fig. 24 The proposed mechanism of phosphoryl group transferred transition state. 54

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