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系統識別號 U0026-0808201215335000
論文名稱(中文) 三環毒素在功能、骨架動態行為與蛋白立體結構上的研究
論文名稱(英文) Functional, dynamic, and structural studies of three-fingered toxins
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 鄭俊和
研究生(英文) Chun-Ho Cheng
學號 s58921183
學位類別 博士
語文別 英文
論文頁數 107頁
口試委員 指導教授-莊偉哲
召集委員-游一龍
口試委員-賴明德
口試委員-周三和
口試委員-陳金榜
中文關鍵字 三環毒素  蛇毒蛋白  骨架動力學  酵母菌表現 
英文關鍵字 dendroaspin  erabutoxin b  muscarinic toxin 2  backbone dynamics  Pichia 
學科別分類
中文摘要 Three-fingered fold的蛋白廣泛分佈在各種組織,並執行各式各樣的功能。這類蛋白通常由60至80個氨基酸組成,且含有4或5個雙硫鍵,兩股和三股的反平行摺板(antiparallel sheet)協助three finger的形成,並使三個loop靠近固定。儘管這個家族的成員結構都很相似,但是其生物性功能歧異性卻很大, Dendroaspin (Den)是由Dendroaspis jamesoni kaimose的毒液中純化出來的three-fingered fold蛇毒蛋白含有PRGDMP motif;Rhodostomin (Rho)是從Calloselasma rhodostoma的毒液中純化出來的蛇毒蛋白屬於去結合蛋白也同樣帶有PRGDMP motif,雖然Den與Rho的立體結構不同,但是一樣有很強integrin抑制力。為了研究結構、功能與骨架動力學的關係,我們藉由Pichia表現系統,生產重組蛋白Den和Rho。純化後Den和Rho的血小版抑制濃度為149.8和83.2 nM,細胞黏著抑制實驗顯示Den比Rho在integrin αIIbβ3弱3.7倍,在integrin αvβ3弱2.5倍,在integrin α5β1結果相似,分別為239.8和256.8 nM。NMR分析顯示Den和Rho在RGD motif有不同的立體構型,但是比較Den和Rho在模擬與integrin αvβ3結合上,有相似的作用數目,但是在骨架動力學Den和Rho表現出不同的特性。這些結果證明蛋白構型會影響RGD motif的功能、結構和骨架動力學,這也說明含RGD蛋白的抑制能力和選擇性,不僅僅單由序列決定而已。為了研究一個相似的蛋白構型可以導致功能差異很大的原因,我們也在酵母菌表現菸鹼受器的antagonist,從海蛇Laticauda semifasciata毒液得到的erabutoxin b 和蕈毒素受器的抑制物,從綠曼巴蛇Dendroaspis angustuceps毒液得到的muscarinic toxin 2 (MT2),最後純化的產量前者約10-15 mg/L,後者約5-10 mg/L。erabutoxin b也經由肌肉神經毒性測試確定其活性。經核磁共振的圖譜分析,確認EBT b與MT2的結構均與原始蛋白相同。分析Den,EBT b和MT2的骨架動力學實驗,顯示他們在loop 1, loop 2和loop 3表現出不同的可動性及不同時間級數的運動。Den只有在含RGD loop 的loop 3可動性最大;相較之下EBT b和Toxin α在loop 1較固定,loop 2和loop 3可動性較大;MT2也是在loop 1和loop 2可動性較大,但是在loop 3可動性較小。比較Den,EBT b和MT2的動力學參數與其他three-fingered 毒素發現,動力學的變異性可能在與不同的目標蛋白在作用上扮演重要的角色。這些結果顯示三環毒素分歧的功能在loop區域的骨架動力中有一定的關連性,這個研究將加強three-fingered fold的結構應用於醫療上的可能性。
英文摘要 Proteins with the three-fingered fold are widely distributed in various tissues where they exert a variety of functions. They comprise 60-80 amino acids with four or five disulfide bonds and are organized into three fingers connected by the double-stranded and triple-stranded antiparallel sheets. Despite the overall similarity in structure, these proteins in this family differ from each other in their biological activities. Dendroaspin (Den) is a three-fingered fold venom protein containing a PRGDMP motif from Dendroaspis jamesoni kaimose and rhodostomin (Rho) is a disintegrin from Calloselasma rhodostoma also with a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris (P. pastoris). The recombinant Den and Rho inhibited platelet aggregation with the KI values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin αIIbβ3 and 2.5 times less active when inhibiting the integrin αvβ3. In contrast, Den and Rho were similarly active when inhibiting the integrin α5β1 with the IC50 values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl-glycyl-aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin αvβ3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif, which suggests that the inhibitory potency and selectivity of RGD-containing proteins cannot be determined only by the sequence content of the RGD loop. To study how a similar fold can accommodate distinct functional topographies, we also expressed erabutoxin b (EBT b), a nicotinic acetylcholine receptor antagonist from Laticauda semifasciata, and muscarinic toxin 2 (MT2), a muscarinic receptor inhibitor from Dendroaspis angusticeps, in P. pastoris. Their yields were 10-15 mg/L and 5-10 mg/L, respectively. EBT b was shown to have muscle-type neuron toxicity. NMR analysis showed that EBT b and MT2 expressed in P. pastoris processes the same structures as those of native proteins. Analysis of the dynamics properties of DEN, EBT b, and MT2 showed that they exhibited difference in the flexibility on the loops I, II, and III and in the motions on multiple time scales. The RGD-containing loop of DEN, loop III, was the most flexible region. In contrast, EBTb and toxin exhibited rigidity on the loop I and flexibility on loops II and III. The loops II and III of MT2 were flexible; however, the loop III was rigid. Comparison of the dynamics properties of DEN, EBTb, and MT2 with other members of three-fingered toxins suggests that their dynamics deviations may play an important role in interacting with different target proteins. These results suggest that divergent functions of three-fingered toxins may be correlated with their backbone dynamics of loop regions.
論文目次 中文摘要 ..……………………………………………………..………………………… i
Abstract ……………………………………………………...................................…..… iii
Acknowledgements ……………………………………………….……….…………..… v
Table of contents …………………………………………………..……………...………vi
List of Tables ………………………………………………………………..………..….. x
List of Figures ……………………………………………………………......………….. xi
Abbreviations ……………………………………………………………..……..………xiii

Chapter 1 Introduction
1.1 Snake toxins ……………………………………………………..……………..…… 1
1.2 Three-fingered toxins ……………………………………………..…………..…….. 1
1.3 Neurotoxins ………………………………………………………..…………..……. 2
1.4 Muscarinic toxins ……………………………………………….….…………..…… 4
1.5 Cardiotoxins ………………………………………………….…….…………..…… 5
1.6 Recent study in three-fingered toxins ……………………….……………………… 5
1.7 Integrins ………………………………………………………..…….……..…..…… 6
1.8 Dendroaspin …………………………………………………..…….…………..…… 7
1.9 Pichia expression system …………………………………….……………………… 8
1.10 Specific Aims …………………………………….…….…………………………… 9
1.10.1 Different scaffolds affect integrin recognition …………………………….…….9
1.10.2 The same scaffold facilitates different recognition functions ………………….10
Chapter 2 Materials and Methods
2.1 Gene design and synthesis ……………………………………………..…….……….11
2.2 Construction of expression vector and transformation …………………………….....11
2.3 Expression of recombinant Den ………………………………………………………12
2.4 Purification of Den by Ni2+-chelating column …………………………………..…....12
2.5 Purification of Den by reverse-phase HPLC …………………………………..……..13
2.6 NMR Spectroscopy …………………………………………………………………...13
2.7 Structure Calculations of Den ……………………………………………………...…14
2.8 Measurements of NMR Dynamics ……………………………………………..……..15
2.9 Molecular docking ……………………………………………………………….……16
2.10 Mass Spectrometry Measurements ……………………………………………..……17
2.11 Platelet Aggregation Assay ……………………………………………………..……18
2.12 Cell Adhesion Assay …………………………………………………………………18

Chapter 3 Results
3.1 Comparison of Den and Rho in function, structure and dynamics
3.1.1 Design and synthesis of Den ……………………………….…….……….…….…..20
3.1.2 Expression and purification of Den ……………………………………….…….…..20
3.1.3 Inhibition of Platelet Aggregation ………………………….…………….……..…..21
3.1.4 Inhibition of Cell Adhesion to Fibrinogen and Fibronectin………………..….…….21
3.1.5 NMR analysis of Den …………………………………………….……..…..………22
3.1.6 Structural determination of Den ……………………………………...…….……….23
3.1.7 Structural difference between the RGD motif of Den and Rho ………...…………..24
3.1.8 Comparison between integrin v3 complexes of Den and Rho …….…..……...…25
3.1.9 Backbone dynamics of Den ……………………………………………...………….25
3.2 Comparison of backbone dynamics of different three-fingered toxins
3.2.1 Backbone dynamics of EBT b ……………………………………………….……...28
3.2.2 Backbone dynamics of MT2 ………………………………………………………..28
3.2.3 Comparison of backbone dynamics among different three-fingered toxins …….….29
3.2.4 Analysis of order parameter (S2) variations ..……………..………………………..30
3.2.5 Analysis of Rex variations …………………..……………..……………………... 30
3.2.6 Analysis of e variations ……………………..…………………………………….30

Chapter 4 Discussion
4.1 Expression strategy ......................................................................................................32
4.2 Functions and mutants of Den .....................................................................................32
4.3 Structrual differences between Den and Rho ...............................................................33
4.4 Den and toxins with similar fold ……………………………….…………………….34
4.5 Comparison of Den and previous Den structure ..........................................................35
4.6 Docking methods ..........................................................................................................36
4.7 Dynamics of Den, EBTb and MT2 ...............................................................................36
4.8 Expression of other toxin and Pichia pastoris ...............................................................38

Chapter 5 Conclusions
Conclusions .........................................................................................................................40
References ...........................................................................................................................41
Tables ...................................................................................................................................48
Figures .................................................................................................................................57
Appendixes ..........................................................................................................................91
A The primers for synthesis of dendroaspin ……………………………………….......91
B Results of the 15N relaxation data and model free parameters
B.1 Experimental 15N Relaxation Data and Optimized Model free Parameters of
Den by 600 MHz NMR ………………………………………………………..92
B.2 Experimental 15N Relaxation Data and Optimized Model free Parameters of
Den by 700 MHz NMR ………………………………………….…………….94
B.3 Experimental 15N Relaxation Data and Optimized Model free Parameters of EBTb by 600 MHz NMR ………..………………………………..…………...96
B.4 Experimental 15N Relaxation Data and Optimized Model free Parameters of EBTb by 800 MHz NMR ………….…………………………..………………98
B.5 Experimental 15N Relaxation Data and Optimized Model free Parameters of
MT2 by 600 MHz NMR ……………………………………………………...100
B.6 Experimental 15N Relaxation Data and Optimized Model free Parameters of
MT2 by 800 MHz NMR …….....……………………………………………..102
C Docking restraints
C.1 The restraints for docking Den to integrin αvβ3 ………………………………104
C,2 Ambiguous interaction restraints for docking Den to integrin αvβ3 ………….107
參考文獻 Antil, S., Servent, D., and Menez. A.: Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of -cobratoxin. J. Biol. Chem. 274: 34851–34858, 1999.
Antil-Delbeke, S., Gaillard, C., Tamiya, T., Corringer, P.J., Changeux, J.P., Servent, D., and Menez, A: Molecular determinants by which a long chain toxin from snake venom interacts with the neuronal alpha 7-nicotinic acetylcholine receptor. J. Biol. Chem. 275: 29594–29601, 2000.
Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., and Kuntz, I.D. Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments. Biochemistry. 27:2763-2771, 1988.
Brunger, A.T. (1992) X-PLOR :version 3.1 : a system for x-ray crystallography and NMR, Yale University Press, New Haven.
Chen, C.Y., Shiu, J.H., Hsieh, Y.H., Liu, Y.C., Chen, Y.C., Chen, Y.C., Jeng, W.Y., Tang, M.J., Lo, S.J., and Chuang, W.J. Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin. Proteins. 76:808-21, 2009.
Chippaux, J.P.: The treatment of snake bites: analysis of requirements and assessments of therapeutic efficacy in tropical Africa. In: Menez A., editor. Perspectives in molecular toxinology. Chichester, England: John Wiley & Sons; p. 457–473, 2002.
Chen, CC. Expression in Pichia pastoris and characterization of erabutoxin b and muscarinic toxin 2, the potent antagonists of acetylcholine receptors. Master thesis of Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, 2006.
Cherny, R.C., Honan, M.A., and Thiagarajan, P. Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion. J. Biol. Chem. 268:9725-9729, 1993..
Chien, K.Y., Chiang, C.M., Hseu, Y.C., Vyas, A.A., Rul,e G.S., and Wu, W. Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions. J Biol Chem. 269:14473-83, 1994.
Dominguez, C., Boelens, R., and Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125:1731-1737, 2003.
Diemand, A.V., and Scheib, H. iMolTalk: an interactive, internet-based protein structure analysis server. Nucleic Acids Res 32 (Web Server issue):W512-516, 2004.
Dryer, S.E., and Chiappinelli, V.A.. Kappa-bungarotoxin: an intracellular study demonstrating blockade of neuronal nicotinic receptors by a snake neurotoxin. Brain Res. 289:317-21, 1983.
Dufton, M.T. and Hider, R.C. Structure and pharmacology of elapid cytotoxins. Pharmacol. Ther. 36: 1–40, 1988.
Endo, T. and Tamiya, N. Structure-function relationships of postsynaptic neurotoxins from snake venoms. In: Harvey A.L., editor. Snake toxins. New York: Pergamon Press; p. 165–222, 1991.
Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N.J., Servent, D. Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and N-methylscopolamine-occupied states. Mol Pharmacol. 74:1554-63, 2008.
Gilquin, B., Bourgoin, M., Nez, R.M., Leneledu, M.H., Servent, D., Zinn-Justin, S., and Menez, A.: Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering. Protein Sci. 12: 266–277, 2003.
Guenneugues, M., Drevet, P., Pinkasfeld, S., Gilquin, B., Menez, A., and Zinn-Justin, S.: Picosecond to hours time scale dynamics of a “ three finger: toxin: correlation with its toxic and antigenic properties. Biochemistry. 36:16097-16108, 1997.
Guo, R.T., Chou, L.J., Chen, Y.C., Chen, C.Y., Pari, K., Jen, C.J., Lo, S.J., Huang, S.L., Lee, C.Y., Chang, T.W., and Chuang, W.J.: Expression of Rhodostomin in Pichia Pastoris and Characterization by Circular Dichroism and NMR of Rhodostomin. Proteins. 43: 499-508, 2001.
Hamilton, S.R., Bobrowicz, P., Bobrowicz, B., Davidson, R.C., Li, H., Mitchell, T., Nett, J.H., Rausch, S., Stadheim, T.A., Wischnewski, H., Wildt, S., and Gerngross, T.U. Production of complex human glycoproteins in yeast. Science. 301:1244-1246, 2003.
Hodgson, W. C. and Wickramaratna, J. C.: In vitro neuromuscular activity of snake venom. Clin. Exp. Pharmacol. Physiol. 29: 807–814, 2002.
Jaseja, M., Luz, X., Williams, J. A., Sutcliffe, M. J., Kakkar, V.V., Parslow, R. A., and Hyde, E. I.: 1H-NMR assignments and secondary structure of dendroaspin, an RGD-containing glycoprotein IIb-IIIa (IIb-3) antagonist with a neurotoxin fold. Eur. J. Biochem. 226: 861-868, 1994.
Joubert, F.J. and Taljaard, N.: Some properties and the complete primary structures of two reduced and S-carboxymethylated polypeptides (S5C1 and S5C10) from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom. Biochim Biophys Acta. 579:228-33, 1979.
Kim, H.S. and Tamiya, N.: Amino acid sequences of two novel longchain neurotoxins from the venom of the sea snake Laticauda colubrina. Biochem. J. 207: 215–223, 1982.
Kini, R. M.: Molecular moulds with multiple missions: Functional sites in three-finger toxins. Clin. Exp. Pharmacol. Physiol. 29: 815–822, 2002.
Krajewski, J.L., Dickerson, I.M., and Potter, L.T. Site-directed mutagenesis of m1-toxin1: two amino acids responsible for stable toxin binding to M(1) muscarinic receptors.Mol Pharmacol. 60:725-31,2001.
Le Goas, R., LaPlante, S.R., Mikou, A., Delsuc, M.A., Guittet, E., Robin, M., Charpentier, I., and Lallemand, J.Y. Alpha-cobratoxin: proton NMR assignments and solution structure. Biochemistry. 31:4867-4875, 1992.
Levandoski, M.M., Caffery, P.M., Rogowski, R.S., Lin, Y., Shi, Q.L., and Hawrot, E.: Recombinant expression of alpha-bungarotoxin in Pichia pastoris facilitates identification of mutant toxins engineered to recognize neuronal nicotinic acetylcholine receptors. J. Neurochem. 74: 1279-1289, 2000.
Liu, Z., Li, W., Zhang, H., Han, Y., and Lai, L.: Modeling the third loop of short-chain snake venom neurotoxins: Roles of the short-range and long-range interactions. Proteins. 42: 6–16, 2001.
Lu, X., Rahman, S., Kakkar, V.V., and Authi, K. S.: Substitutions of proline 42 to alanine and methionine 46 to asparagine around the RGD domain of the neurotoxin dendroaspin alter its preferential antagonism to that resembling the disintegrin elegantin. J. Biol. Chem. 271: 289–294, 1996.
Lu, X., Sun Y., Shang D., Wattam B., Egglezou, S., Hughes, T., Hyde, E., Scully, M. and Kakkar, V.: Evaluation of the role of proline residues flanking the RGD motif of dendroaspin, an inhibitor of platelet aggregation and cell adhesion. Biochem. J. 355: 633-638, 2001.
McDowell, R.S., Dennis, M.S., Louie, A., Shuster, M., Mulkerrin, M.G., and Lazarus, R.A.: Mambin, a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor structurally related to the short neurotoxins. Biochemistry. 31: 4766-4772, 1992.
Menez, A.: Functional architectures of animal toxins: a clue to drug design? Toxicon. 36:1557–1572, 1998.
Nirthanan, S. and Gwee, M.C.E.: Three-finger neurotoxins and the nicotinic acetylcholine receptor. J. Pharmacol. Sci. 94: 1 – 17, 2004.
Nirthanan, S., Gopalakrishnakone, P., Gwee, M.C.E., Khoo, H.E., and Kini, R.M.: Non-conventional toxins from Elapid venoms. Toxicon. 41:397–407, 2003.
Osipov, A.V., Rucktooa, P., Kasheverov, I.E., Filkin, S.Y., Starkov, V.G., Andreeva, T.V., Sixma, T.K., Bertrand, D., Utkin, Y.N., and Tsetlin, V.I. Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors. J. Biol. Chem. 287:6725-34, 2012.
Pfaff, M., McLane, M.A., Beviglia, L., Niewiarowski, S., Timpl, R. Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition. Cell Adhes Commun.2:491-501, 1994.
Pawlak, J., Mackessy, S.P, Fry, B.G., Bhatia, M., Mourier, G., Fruchart-Gaillard, C., Servent, D., Menez, R., Stura, E., Menez, A., and Kini, R.M. Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity. J. Biol. Chem. 281:29030-29041, 2006.
Pawlak, J., Mackessy, S.P., Sixberry, N.M., Stura, E.A., Le Du, M.H., Menez, R., Foo, C.S., Menez, A., Nirthanan, S., and Kini, R.M. Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity. FASEB J. 23:534-45, 2009.
Rahman, S., Aitken, A., Flynn, G., Formstone, C., Savidge, G.F. Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation. Biochem J 335:247-257, 1998.
Roy, A., Zhou, X., Chong, M.Z., D'hoedt, D., Foo, C.S., Rajagopalan, N., Nirthanan, S., Bertrand, D., Sivaraman, J., and Kini, R.M. Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra). J. Biol. Chem. 285:8302-15, 2010.
Samson, A.O., Chill, J.H., and Anglister, J. Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide. Biochemistry. 44:10926-10934, 2005.
De Schutter, K., Lin, Y.C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouze, P., Van de Peer, Y., and Callewaert, N. Genome sequence of the recombinant protein production host Pichia pastoris. Nat. Biotechnol. 27:561-566, 2009.
Shiu, J.H., Chen, C.Y., Chang, L.S., Chen, Y.C., Chen, Y.C., Lo, Y.H., Liu, Y.C., and Chuang, W.J.: Solution structure of -bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding. Proteins. 57: 839-849, 2004.
Shiu, J.H., Chen, C.Y., Chen, Y.C., Chang, Y.T., Chang, Y.S., Huang, C.H., and Chuang, W.J. Effect of P to A mutation of the N-terminal residue adjacent to the Rgd motif on rhodostomin: importance of dynamics in integrin recognition. PLoS One. 7:e28833, 2012.
Sue, S.C., Rajan, P.K., Chen, T.S., Hsieh, C.H., and Wu, W. Action of Taiwan cobra cardiotoxin on membranes: binding modes of a beta-sheet polypeptide with phosphatidylcholine bilayers. Biochemistry. 36:9826-36, 1997.
Sutcliffe, M.J., Jaseja, M., Hyde, E. I., Lu, X., and Williams, J.A.: Three-dimensional structure of the RGD-containing neurotoxin homologue dendroaspin. Nat. Struct. Biol. 1: 802-7, 1994.
Takacs, Z., Wilhelmsen, K.C., and Sorota, S.: Snake a-neurotoxin binding site on the Egyptian cobra (Naja haje) nicotinic acetylcholine receptor is conserved. Mol. Biol. Evol. 18:1800–1809, 2001.
Takagi, J., Kamata, T., Meredith, J., Puzon-McLaughlin, W., and Takada, Y. Changing ligand specificities of alphavbeta1 and alphavbeta3 integrins by swapping a short diverse sequence of the beta subunit. J Biol Chem 272:19794-19800, 1997.
Tsetlina,V. I. and Huchob , F.: Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications. FEBS Letters. 557: 9-13, 2004.
Tsetlin, V.: Snake venom a-neurotoxins and other `three-finger' proteins. Eur. J. Biochem. 264: 281-286, 1999.
Wattam, B., Shang, D., Rahman,, S., Egglezou, S., Scully, M., Kakkar, V., and Lu, X.: Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of 1 and 3 integrins. Biochem. J. 356: 11-17, 2001.
Xiong, J.P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., and Arnaout, M.A. Crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with an Arg-Gly-Asp ligand. Science 296:151-155, 2002.
Zhang, X.P., Kamata, T., Yokoyama, K., Puzon-McLaughlin, W., and Takada, Y. Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin alphavbeta3. J Biol Chem 273:7345-7350, 1998.
Zinn-Justin, S., Roumestand, C., Gilquin, B., Bontems, F., Ménez, A., and Toma, F. Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study. Biochemistry. 31:11335-11347, 1992.
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