臺灣博碩士論文加值系統

具細胞毒性之核醣核酸水解酶與肝素
複合物晶體結構之研究
摘要
牛蛙的RNA水解酶與RNase A屬於同一個家族，牛蛙的RNA水解酶除了水解RNA的催化功能以外，還帶有細胞毒性、外源凝集素及黏著到癌細胞表面的特性。為了進一步從結構生物學的角度了解蛙類RNA水解酶外源凝集素的特性，我們利用牛蛙其中一種RNA水解酶RC-RNase3，得到RC-RNase3與肝素雙醣複合物的晶體，肝素是一種葡萄糖胺聚合糖，常見於細胞表面或細胞外間質。RC-RNase3和肝素的複合物的結構是由七個β-strand和三個α-helix組成，形成一個碗狀。我們在RC-RNase3的sheet 1 (β1, β2, β4, β5) 和sheet 2(β3, β6, β7)之間發現肝素的位置。肝素其中的一個硫酸根和非催化性鍵結的RC-RNase6和d(CpG)複合物中的硫酸根相當接近，另外肝素其中兩個硫酸根和催化性鍵結的RC-RNase和d(ApCpGpA)複合物中的磷酸根的位置也很接近，我們的結果顯示肝素和上述兩種複合物使用類似的鍵結位置和環境。
台灣雨傘節之類毒蕈鹼毒素BM14及BM8
X光繞射分析
摘要
從台灣雨傘結的蛇毒中分離出兩個新的類毒蕈鹼毒素蛋白質，BM14及BM8均為82個胺基酸的蛋白質，它們的不同在於第37及38個胺基酸的部份，BM14是Lys37-Lys38，BM8是Glu37-Ala38。和BM8不同，BM14表現出與M2型的毒蕈鹼乙醯膽鹼受器有結合能力，這顯示了BM14中的Lys37-Lys38在鍵結上扮演了重要的角色。雖然BM14及BM8和蛇毒心臟毒素有著20~38%的一級序列相似度，卻無細胞毒性。BM14和BM8中10個Cys中的8個與神經毒素、心臟毒素及毒蕈鹼毒素的序列比對中位於保留位置。我們利用結晶的方法來研究其結構，分別得到了BM14及BM8的晶體，BM14的晶體繞射解析度為3.0Å且屬於tetragonal晶系I422空間群，晶胞參數為a=81.68Å、b=81.68Å、c=207.30Å、α=β=γ=90°， BM8的晶體繞射解析度為3.8 Å且屬於hexagonal晶系，其晶胞參數為 a=55.37Å、b=55.37Å、c=338.0Å、α=β=90°、γ=120°。

Crystal Structure of Cytotoxic RC-RNase3 from
Rana catesbeiana in Complex with Heparin
Abstract
The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity and lectin activity. RC-RNase3 has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. The crystal structure of RC-RNase3 in complex with heparin disaccharide was determined to resolution of 1.6Å. Heparin is one type of glycosaminoglycan locating primarily on the surface of cells or in the extra-cellular matrix. The overall structure of RC-RNase3-heparin complex consists of seven β-strands and three α-helices that present a bowl shape. Heparin was found locating between sheet 1 (β1, β2, β4, and β5) and sheet 2 (β3, β6, and β7). One of the sulfate groups in heparin takes place in a very close position to the free sulfate in the retro binding RC-RNase6 and d(CpG) complex. More interestingly, two sulfate groups in heparin are close to the phosphate groups of d(ApCpGpA) in the catalytic binding RC-RNase and d(ApCpGpA) complex. Our results indicate a new binding mode where heparin sharing approximately the same binding site to the catalytic and retro binding modes.
Crystallization and Preliminary X-ray Diffraction Analysis of Muscarinic Toxin-Like Protein,
BM14 and BM8 from Bungarus multicinctus
Abstract
Two novel proteins BM14 and BM8 were isolated from Bungarus multicinctus (Taiwan banded krait) venom. BM14 and BM8 are sequence identical except Lys37-Lys38 in BM14 and Glu37-Ala38 in BM8. In contrast to BM8, BM14 exhibited an activity on binding to the M2 muscarinic acetlycholine (mAchR) receptor subtype, indicating Lys37 and Lys38 of BM14 play the crucial roles on binding to M2 mAchR. Although BM14 and BM8 shared a 20-38% sequence identity with snake venom cardiotoxins, they did not show the cytotoxicity. Eight out of ten Cys residues in BM14 and BM8 are located at the conserved positions as those in neurotoxin, cardiotoxin and muscarinic toxin protein sequences, so called the three-finger protein. BM14 and BM8 crystals were grown by the hanging drop vapor diffusion method. The crystals of BM14 diffract to 3.0Å and belong to the tetragonal space group I422 with unit-cell parameters a=81.68Å, b=81.68Å, c=207.30Å, and α=β=γ=90°. The crystals of BM8 diffract to 3.8 Å and belong to the hexagonal space group with unit-cell parameters a=55.37Å, b=55.37Å, c=338.0Å, α=β=90°, and γ=120°.

Contents
Part I
Chapter 1 Introduction
1.1 Preface 1
1.2 RNase A 2
1.3 Ribonuclease Inhibitor 3
1.4 Onconase 4
1.5 Rana catesbeiana Ribonucleases 5
1.6 Base Specificity 6
1.7 Structural Stability 7
1.8 Cytotoxicity, Antitumor Activity, and Lectin Activity 8
1.9 Glycosaminoglycans 9
Chapter 2 Materials and Methods
2.1 Purification and Crystallization 10
2.1 Data Collection and Process 10
2.3 Data Evaluation 11
2.4 Molecular Replacement by AmoRe 12
2.5 Model Building and Refinement 13
Chapter 3 Results
3.1 Data Statistics and Space Group Determination 14
3.2 Self Rotation 15
3.3 Structure Determination 15
3.4 Model building and Refinement 15
3.5 Crystal Structure of RC-RNase3-heparin complex 16
Chapter 4 Discussion
4.1 Different Binding Mode of RC-Rnases 17
4.2 Superimposition Different RC-RNase complexes 17
4.3 Superimposition of Lgands from
Different RC-RNase Complexe 18
Figures and Legends 19
Tables 33
Reference 37
Part II
Chapter 1 Introduction
1.1 Preface 40
1.2 BM14 and BM8 42
Chapter 2 Materials and Methods
2.1 Sample preparation 43
2.2 Crystallization 43
2.3 Data Collection and Data Process 43
Chapter 3 Results
3.1 Space group determination of BM14 45
3.2 Space group determination of BM8 46
Chapter 4 Discussion
4.1 The extra long loop II 47
4.2 Future Works 47
Figures and Legends 48
Tables 57
Reference 61

Aguilar, C. F., Thomas, P., Moss, D., Mills, A., Palmer, R. A. (1991). "Novel nonproductively bound ribonuclease inhibitor complexes -- high resolution X-ray refinement studies on the binding of RNase A to cytidylyl-2',5'guanosine (2',5'CpG) and deoxycytidylyl- 3',5'-guanosine (3',5'CpdG)." Biochem. Biophys. Acta 1118: 6-20.
Ardelt, W., Mikulski, S. M., Shogen, K. (1991). "Amino acid sequence of an anti-tumor protein from Ranapipiens oocytes and early embryos." J. Biol. Chem. 266: 245-251.
Boix, E., Wu, Y., Vasandani, V. M., Saxena, S. K., Ardelt, W., Ladner, J., Youle, R. J. (1996). "Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity." J. Mol. Biol. 257: 992-1007.
Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, N., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., Warren, G. L. (1998). "Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination." Acta Cryst. D54: 905-921.
Cafaro V., Bracale A., Di Maro A., Sorrentino S., D'Alessio G., Di Donato A. (1998). "New muteins of RNase A with enhanced antitumor action." FEBS Letters, 437: 149-152.
D'Alessio, G. R., J. F. (1997). Ribonuclease: Structures and Functions. San Diego, Academic Press.
Fedorov A. A., J.-M. D., Fedorov E., Sirakova D., Graf I., Almo S. C. (1996). "Ionic interactions in crystalline bovine pancreatic ribonuclease A." Biochemistry 35: 15962-15979.
Irie, M., Nitta, K., Nonaka, T. (1998). "Biochemistry of frog ribonucleases." Cell. Mol. Life Sci. 54: 775-784.
Laskowski, R. A., MacArthur, M. W., Moss, D., Thornton, J. M. (1993). "PROCHECK: a program to check the stereochemical quality of protein structures." J. Appl. Crystallog. 26: 283-291.
Lee F. S., Vallee, B. L. (1993). "Structure and action of mammalian ribonuclease (angiogenin) inhibitor." Prog. Nuvleic Acid Res. Mol. Biol. 44: 1-30.
Leland P. A., Staniszewski, K. E., Kim B.-M., Raines R. T. (2000). "A synapomorphic disulfide bond is critical for the conformational stability and cytotoxicity of an amphibian ribonuclease." FEBS Lett. 477: 203-207.
Leland, P. A., Schultz, L., Kim, B. M., Raines, R. T. (1998). "Ribonuclease A variants with potent cytotoxic activity." Proc. Natl Acad. Sci. USA, 95: 10407-10412.
Leland, P. A. Raines, R. T. (2001). "Cancer chemotherapy-ribonucleases to the rescue." Chem. Biol. 8: 405-413.
Leu, Y. J., Chern,S. S., Wang, S. C., Hsiao, Y. Y., Amiraslanov, I., Liaw, Y. C., Liao, Y. D. (2003). "Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeiana Based upon Mutagenesis and X-ray Crystallography." J. Biol. Chem. 278(9): 7300-7309.
Liao, Y. D., Huang, H. C., Leu, Y. J., Wei, C. W., Tang, P. C., Wang, S. C. (2000). "Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)." Nucleic Acids Res. 28: 4097-4104.
Liu, J. H., Liao, Y. D., Sun, Y. J. (2001). "Crystallization and preliminary X-ray diffraction analysis of cytotoxic ribonucleases from bullfrog Rana catesbeiana." Acta Cryst. D57: 1697-1699.
Lubert Stryer (1994) "Chapter18: Carbohydrates" Stryer's Biochemistry
Matthews, B. W. (1968). "Solvent content of protein crystals." J. Mol. Biol. 33: 491-497.
McRee, D. E. (1999). "XtalView/Xfit-A Versatile Program for Manipulating Atomic Coordinates and Electron Density." J. Struct. Biol. 125: 156-165.
Navaza, J. (1994). "AMoRe: an Automated Package for Molecular Replacement." Acta Cryst. A50: 157-163.
Nitta, K., Ozaki, K., Ishikawa, M., Furusawa, S., Hosono, M., Kawauchi, H., Sasaki, K., Takayanagi, Y., Tsuiki, S., Hakomori, S. (1994). "Inhibition of cell proliferation by Rana catesbeiana and R. japonica lectins belonging to the ribonuclease superfamily." Cancer Res. 54: 920-927.
Okabe, Y., Katayama, N., Iwama, M., Watanabe, H., Ohgi, K., Irie, M., Nitta, K., Kawauchi, H., Takayanagi, Y., Oyama, F. (1991). "Comparative base specificity, stability, and lectin activity of two lectins from eggs of Rana catesbeiana and R. japonica and liver ribonuclease from R. catesbeiana." J. Biochem. 109: 786-790.
Otwinowski, Z., Minor, W. (1997). "Processing of X-ray Diffraction Data Collected in Oscillation Mode." Methods Enzymol. 276: 307-326.
Vitagliano, L., Merlino, A., Zagari, A., Mazzarella, L. (2000). "Productive and nonproductive binding to ribonuclease A: X-ray structure of two complexes with uridylyl(2',5')guanosine." Protein Sci. 9: 1217-1225.
Aird, S. D., Womble, G. C., Yates III, J. R., Griffin, P. R. (1999). "Primary structure of gamma-bungarotoxin, a new postsynaptic neurotoxin from the venom of Bungarus multicinctus." Toxicon 37: 609-625.
Chang, L. S., Lin, S. R., Wang, J. J., Hu, W. P., Wu, B. N., Huang, H. B. (2000). "Structure-function studies on Taiwan cobra long neurotoxin homolog." Biochim. Biophys. Acta. 1480: 293-301.
Chiappinelli, V. A. (1991). "κ-Neurotoxins and α-neurotoxins: effects on neuronal nicotinic acetylcholine receptors. In: Snake Toxins, Harvey, A.L. ed." New York, USA: Pergamon Press 223-258.
Chung, C., Wu, B. N., Yang, C. C., Chang, L. S. (2002). "Muscarinic Toxin-like Proteins from Taiwan Banded Krait (Bungarus multicinctus) Venom: purification, Characterization and Gene Organization." Biol. Chem. 383: 1397-1406.
Dewan, J. C., Grant, G. A., Sacchettini, J. C. (1994). "Crystal structure of κ-bungarotoxin at 2.3 resolution." Biochemistry 33: 13147-13154.
Endo, T., Tamiya, N. (1991). "Structure-function relationship of postsynaptic neurotoxins from snake venoms. In: Snake Toxins, (Harvey, A. L., ed." New York, USA: Pergamon Press: 165-222.
Harel, M., Kleywegy, G. J., Ravelli, R. B., Silman, I., Sussman, J. L. (1995). "Crystal structure of an acethylcholinesterase-fasciculin complex: interaction of a three-finger toxin from snake venom with its target." Structure 3: 1355-1366.
Love, R. V., Stroud, R. M. (1986). "The crystal structure of alpha-bungarotoxin at 2.5 resolution: relation to solution structure and binding to acetylcholine receptor." Protein Eng. 1: 37-46.
Menez, A. (1998). "Functional architectures of animal toxins: a clue to drug design?" Toxicon 36: 1557-1572.
Ohno, M., Menez, R., Ogawa, T., Danse, J. M., Shimohigashi, Y., Fromen, C., Ducancel, F., Zinn-Justin, S., Le Du, M. H., Boulain, J. C., Tamiya, T., Menez, A. (1998). "Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?" Prog. Nucleic Acid Res. Mol. Biol. 59: 307-364.
Qian, Y. C., Fan, C. Y., Gong, Y., Yang, S. L. (1999). "cDNA sequence analysis of a novel member of the three loop protein family from the Chinese continental banded krait." Biosci. Biotechnol. Biochem. 63: 940-942.
Tsetlin, V. I. (1999). "Snake venom alpha-heurotoxins and other "three-finger" proteins." Eur. J. Biochem. 264(281-286).
Yu, C., Bhaskaran, R., Chung, L. C., Yang, C. C. (1993). "Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study." Biochemistry 32(2123-2136).