Structural Studies of Human RNase 7 and lebsiella pneumoniae Phosphatase Wzb

第一章醣核酸水解酵素廣泛的存在各種生物體中,並且在核醣核酸的代謝,血管新生,神經毒性,抗癌和抗菌等活性上扮演著重要的角色。其中在人類核醣核酸水解酵素中,核醣核酸水解酵素第三號,第五號及第七號蛋白具有抗菌活性。雖然這些酵素都具有一些相似的特性,例如高等電位點及淨正電荷,但是它們的抗菌機制仍然未明。探討帶有正電荷的胺基酸或決定位對抗菌活性是否有相關性的研究是非常有趣的。因此,我們的研究是關於人類核醣核酸水解酵素第七號蛋白上帶有正電荷性的胺基酸在抗菌中扮演的角色。我們發現人類核醣核酸水解酵素第七號蛋白在酸鹼值3.5-9.5之間都很穩定,尤其二級結構對於溫度(Tm值66.4 度)和化學物質GldnHCl (Cm值3.27 M)也相當穩定。人類核醣核酸水解酵素第七號蛋白的核磁共振(NMR)結構研究顯示這22個帶有正電荷胺基酸(其中18個是離胺酸,4個是精胺酸)主要是分布於蛋白表面的三個區域。第一個區域包含離胺酸1/離胺酸3和離胺酸111/離胺酸112,是相當的具有易彎曲性,並且座落在蛋白N端部分;相對的另外兩組是離胺酸32/離胺酸35和離胺酸96/精胺酸97/離胺酸100,則是座落在構形比較固定的二級結構的區域上。突變實驗顯示,在N端易彎曲部位的正電荷胺基酸相對於催化性胺基酸,組胺酸15/離胺酸38/組胺酸123,以及另外兩組正電荷群在抗菌活性上是較為重要的。因此,我們猜測此蛋白可能藉由其N端易彎曲部位上的離胺酸1/離胺酸3和離胺酸111/離胺酸112與細菌的細胞膜作結合,進而改變細胞膜的滲透性。二章細菌中的低分子量蛋白質酪氨酸去磷酸化酵素是一種廣泛的酪氨酸去磷酸化的調控子。儘管它們大量地存在,但是其在原核生物中的功能仍然未闡明。一些研究顯示此蛋白與莢膜多醣體/胞外多醣的合成和輸出及壓力抵抗是有相關。我們的目標蛋白: 酪氨酸去磷酸化酵素是存在於自台灣大學附設醫院臨床上分離出來克雷白氏肺炎菌中。此蛋白具有與其它酪氨酸去磷酸化酵素相同的特徵:如CX5RS序列,存在於已知的P loop或PTPloop;及獨特位於此蛋白的DPY-loop上的的天門冬氨酸。我們之前的研究顯示將此蛋白上的第九顆半胱氨酸突變成絲胺酸會使其失去酵素活性。為了確定此類蛋白上保留的第九顆半胱氨酸在催化機制的重要性,我們表現和純化未標示及同位素標示的酪氨酸去磷酸化酵素及C9S突變蛋白去做結構分析。由於好幾顆胺基酸在酪氨酸去磷酸化酵素的核磁共振光譜上消失了,我們因而不能決定其結構。相較之下,C9S突變蛋白有較好品質的核磁共振光譜,而它具有與一般低分子量蛋白質酪氨酸去磷酸化酵素相同的摺疊方式,包含在中間由四個平行的的beta-摺板及五個alpha-螺旋的三維結構已被我們解出。將此酪氨酸去磷酸化酵素及C9S突變蛋白的HSQC光譜重疊發現在酪氨酸去磷酸化酵素的光譜上消失及移動的訊號幾乎都是座落在P-loop上及其附近的區域。蛋白骨幹(backbone)的動態研究顯示在P-loop區域的胺基酸是中度穩固。除此之外,突變蛋白的溫度熔點和化學熔點都是高於酪氨酸去磷酸化酵素。因此,我們推斷P-loop在酪氨酸去磷酸化酵素和C9S突變蛋白中有構形上的變化,P-loop在C9S突變蛋白上顯然是較為穩固的。由於P-loop穩固性的改變,導致絲胺酸在C9S突變蛋白中不能像半胱氨酸一樣做為親核劑去攻擊磷酸根,進而使酵素失去活性。Chapter 1ibonucleases are found widely within living organisms and are thought to play important roles in RNA metabolism, angiogenesis, neurotoxicity, antitumor and antimicrobial activity. In human ribonucleases, antimicrobial activity was found in RNase 3, RNase 5 and RNase 7. The mechanisms of antimicrobial activities remain unclear although they possess similar properties, e.g., high isoelectric point and net positive charge. It is therefore interesting to investigate whether the positively charged residues or epitopes are responsible for the bactericidal activity. Here, we report on the role of cationic residues of human RNase 7 (hRNase 7) in its antimicrobial activity. We found that hRNase 7 is stable in the range of pH 3.5~9.5 and its structure is thermally and chemically stable with a Tm value of 66.4 ℃ and a Cm value of 3.27 M denatured by guanine hydrochloride. NMR structure of hRNase 7 shows that the 22 positively charged residues (18 Lys, 4 Arg) are distributed into three clusters on the surface. Residues in the first cluster including Lys1, Lys3, Lys111 and Lys112, are quite flexible and located near the N-terminus. On the contrary, the other two clusters, one with residues Lys32/Lys35 and the other with Lys96/Arg97/Lys100, are all located on secondary structure regions and are quite rigid. Mutagenesis studies confirmed that residues in the flexible cluster are critical for the bactericidal activity rather than the catalytic residues, such as His15, Lys38, His123, and residues in the other two positively charged clusters. Together, we suggest that hRNase 7 binds to bacterial membrane primarily by using the flexible residues, Lys1, Lys3, Lys111, and Lys112, in the first positively charged cluster and then renders the membrane permeable.hapter 2he low molecular weight protein tyrosine phosphatases (LMW-PTPs) in bacteria are ubiquitous regulators of tyrosine phosphorylation. Despite their abundance, the spectrum of functions of LMW-PTPs in prokaryote has yet to be elucidated. Several studies have demonstrated a role of LMW-PTPs in CPS/EPS synthesis and export, as well as in stress resistance. The target protein, LMW-PTPs Wzb, studied in this thesis is from the K. pneumoniae NTUH2044 strain clinically isolated at National Taiwan University Hospital (NTUH). It contains the conserved signature motif of CX5RS known as the P-loop or PTPloop, and an unique Asp residue situated on the DPY-loop of the LMW-PTPs. Our previous study has found that catalytic activity on Wzb_C9S mutant is dramatically reduced or even not seen at all. To ascertain the importance of the conversed residue Cys9 in the catalytic mechanism of Wzb, we expressed and purified unlabeled and isotopically labeled Wzb and Wzb_C9S mutant for structural study. Due to the lack of several cross peaks in NMR spectral of Wzb, we could not determine its structure. In comparison, Wzb_C9S gave much better NMR quality and its 3D NMR solution structure containing a canonical topology of LMW-PTPs with a central four-stranded parallel beta-sheet and five alpha-helices has been solved. Superimposition of 2D HSQC spectra of Wzb and Wzb_C9S reveals that the unobserved cross peaks and cross peaks exhibiting shift changes in Wzb are mostly located in the P-loop and its nearby regions. Backbone dynamics study showed that the residues in the P-loop are moderately rigid. In addition, both Tm and Cm values in Wzb_C9S are higher than those in Wzb. Taken together, we conclude there is a conformational change, especially in the P-loop, between Wzb and Wzb_C9S, with the P-loop in Wzb_C9S apparently more rigid. Due to the change of the rigidity in the P-loop, the Ser9 in C9S mutant is unable to act as a nucleophile to attack the phosphate group, as Cys9 does in Wzb.口試委員審定書 i誌 ii文摘要 iiibstract vist of Abbreviations ixist of Figures xiiist of Tables xiv hapter 1 The flexible and clustered lys residues is critical for antimicrobial activity of human RNase 7.1 Introduction 2-1-1 Antimicrobial peptide/protein diversity 2-1-2 Mechanisms of antimicrobial peptides/proteins 3-1-3 Introduction to human RNase 7 and other RNase family 4.2 Materials and Methods 7.2.1 Preparation of human RNase 7 7.2.2 Circular dichroism (CD) experiments 7.2.3 NMR spectroscopy 8.2.4 Hydrogen bonds and disulfide restraints 8.2.5 NMR restraints and tertiary structure calculation9.2.6 Relaxation measurements 9.2.7 Model-free analysis 10.3 Result 11.3.1 Basic property of human RNase 7 11.3.2 Conformational and structural stability of human RNase 7 11.3.3 Structure description of human RNase 7 12.3.4 Residues responsible for bactericidal activity of human RNase 7 13.3.5 Structural comparison of wild type and K3A mutated hRNase 7 14.3.6 Backbone dynamics analysis 14.4 Discussion 15hapter 2 Solution structural studies of Klebsiella pneumoniae phosphatase Wzb.1 Introduction 31.1.1 Klebsiella pneumoniae and CPS/LPS formation 31.1.2 Phosphatases and kinases in bacteria 32.1.3 Phosphatase Wzb from Klebsiella pneumoniae 35.2 Material and Methods 41.2.1 Preparation of K. pneumoniae protein-tyrosine phosphatase (Wzb) 41.2.2 Assay for protein tyrosine phosphatase activity 41.2.3 Circular dichroism (CD) experiment 42.2.4 NMR experiments and resonance assignments 42.2.5 H/D exchange 43.2.6 Structure calculation and analysis 43.2.7 Relaxation measurements 44.3 Result 46.3.1 Basic property of Wzb 46.3.2 Conformational and structural stability of Wzb and Wzb_C9S 46.3.3 Structure description of Wzb_C9S 47.3.4 Structural comparison of Wzb and Wzb_C9S 48.3.5 Backbone dynamics analysis of Wzb and Wzb_C9S 49.4 Discussion 50eferences 67ppendix 7