整合蛋白(integrin)是一群由α及β次單元體所形成的異構穿膜雙體蛋白，它們表現在細胞的表面上並賦予細胞黏著能力與媒介細胞與細胞或細胞與細胞間質的交互作用。由於整合蛋白的異常與許多疾病的發生有關，包括腫瘤新生、癌細胞轉移、骨質疏鬆症及凝血功能不全症，突顯了整合蛋白作為藥物設計的目標。去整合蛋白則是一群在蛇毒液所發現的強效整合蛋白抑制劑。馬來蝮蛇蛇毒蛋白(Rhodostomin, Rho)屬於去整合蛋白中的一員，具有48PRGDMP辨識序列、連接區域為39SRAGKICRI序列功能區與C端為65PRYH序列並形成6對雙硫鍵。在本篇研究中，我使用馬來蝮蛇蛇毒蛋白當作骨架來研究去整合蛋白與整合蛋白間的辨識機制。藉由運用細胞黏著競爭實驗、核磁共振圖譜(NMR)、X射線結晶學與分子嵌合方法，來探討以下4個關於去整合蛋白與整合蛋白之間結構與功能性相關性的研究主題: (1)含有48ARGDWN序列馬來蝮蛇蛇毒蛋白與其C端區域能互相協同並調控與整合蛋白αIIbβ3之間的辨識。為了瞭解RGD loop與C端區域在去整合蛋白所扮演的角色，我們利用酵母菌產製了2種RGD序列(48PRGDMP和48ARGDWN)搭配5種C端區域序列(65P、65PR、65PRYH、65PRNGLYG和65PRNPWNG)組合的馬來蝮蛇蛇毒突變蛋白。C端區域對於整合蛋白的影響程度為αIIbβ3 > αVβ3 > α5β1。48ARGDWN-65PRNPWNG突變蛋白具有最高辨識整合蛋白αIIbβ3的選擇性;然而，48PRGDMP-65PRNPWNG突變蛋白則不具有任何整合蛋白的選擇性。NMR結構分析48ARGDWN-65PRYH、48ARGDWN-65PRNGLYG與48ARGDWN-65PRNPWNG突變蛋白證實了他們的C端區域與RGD loop進行交互作用，尤其是他們的W52胺基酸分別與65PRYH上的H68胺基酸、65PRNGLYG上的L69胺基酸與65PRNPWNG上的N70胺基酸進行交互作用。在48ARGDWN-65PRNPWNG與整合蛋白αIIbβ3電腦嵌合分析中也發現胺基酸N70能與αIIb上的D159胺基酸形成氫鍵，胺基酸W69能與β3上的K125胺基酸形成cation-pi作用力。我們的結果說明了去整合蛋白的RGD loop與C端區域能夠進行協同作用進而影響其結構與對整合蛋白的辨識; (2) 48ARGDDP突變蛋白對於整合蛋白αVβ3之間的選擇性抑制機制。為了瞭解胺基酸X在ARGDX序列中扮演的角色，我們產製了一系列的48ARGD52XP突變蛋白。X52胺基酸對於整合蛋白的影響程度為α5β1 (86-fold) > αIIbβ3 (41-fold) > αVβ3 (14-fold)。其中48ARGDDP突變蛋白能專一性辨識整合蛋白αVβ3，其抑制整合蛋白αVβ3、αIIbβ3、αVβ5、αVβ6與α5β1的IC50數值分別為45.3、5117.2、6886、14980.3與5044.5 nM。X-ray結構分析證明了Arg49與Asp52胺基酸之間的Cα原子距離為5.9 Å，與目前已知能專一性抑制整合蛋白αVβ3的結構需求條件一致。M52D突變也導致負電區域出現，推測與下降抑制整合蛋白αIIbβ3、αVβ5、αVβ6與α5β1活性相關。而在體外試驗也證明48ARGDDP突變蛋白仍保有抑制內皮細胞的爬行與血管生成作用，說明了此突變蛋白應用於抑制血管增生的潛力; (3) 48ARGDPP突變蛋白喪失與整合蛋白之間辨識的機制。48ARGDPP是個沒有活性的突變蛋白，其抑制整合蛋白αVβ3、αIIbβ3、αVβ5、αVβ6與α5β1的IC50數值分別為41260、64665、35247、15055.3與62460 nM。X-ray結構分析發現48ARGDPP突變蛋白具有兩種構型與P52突變導致了RGD區域結構的改變。位置52的Met突變成Pro導致構型A上的D51-P52之間的胜肽鍵與構型B上的P52-P53之間的胜肽鍵產生反式構型。而在胺基酸R49與P52兩個Cα之間的距離增加成8.0 Å，說明了RGDX功能域(motif)的β轉折結構(turn)的破壞。接著，藉由拉氏圖(Ramachandran plot)分析發現胺基酸D51已經轉變成延展的構型，並導致RGDX功能域的失活。我們的結果也同樣證實了RGDX胜肽上的β轉折結構的重要性; (4) 39KKARTICAR-48GRGDNP-65PRYH (KG) 與39KKARTICAR-48GRGDNP-65PGLYG (KG-P)突變蛋白下降對於抑制整合蛋白αIIbβ3活性的機制。KG與KG-P突變蛋白相較於Rho而言，大大的降低了對於抑制血小板凝集的活性，分別為56與384倍的下降。X-ray結構分析同樣發現他們的C端區域會與RGD loop進行交互作用，例如在KG突變蛋白上可以發現R56胺基酸與Y67胺基酸形成氫鍵; 在KG-P突變蛋白上可以發現D55胺基酸與L67與Y68胺基酸形成氫鍵，R49與N52胺基酸與Y68胺基酸形成氫鍵。它們具有較窄的RGD loop，表面則是呈現不同的電荷分布。從蛋白交互作用嵌合程式分析中我們則發現了此由氨基酸Arg46與Arg66所形成的正電區塊會與整合蛋白αIIb上insert-3區域的D159胺基酸形成鹽橋，在其他α次單元上則具有較短的insert-3而無法曝露出來與正電區塊作用。我們回去觀察野生型Rho的表面電荷分布，發現氨基酸Arg46與Arg66在立體結構上是聚集且形成正電區塊的，可是相較於KG與KG-P則發現正電區塊由於突變的關係而逐漸消失。我們的結果說明了去整合蛋白的氨基酸Arg46與Arg66所形成的正電區塊對於辨識整合蛋白αIIb上insert-3區域的負電區塊扮演重要的角色。我們的結果說明了去整合蛋白的RGD loop、連接區域與C端區域能夠進行協同作用進而影響其結構與對整合蛋白的辨識。總結來說，藉由解答去整合蛋白與整合蛋白結構與功能之間的相關性，加速連結了我們的基礎研究到臨床藥物的開發。

Integrins are αβ heterodimeric receptors that mediate cell-cell and cell-extracellular matrix interactions. Because integrins are involved in tumor progression, thrombosis, and osteoporosis, they are important therapeutic targets. Disintegrins are a family of potent integrin inhibitors that found in snake venoms. Rhodostomin (Rho) is a disintegrin containing a 48PRGDMP motif, a 39SRAGKICRI linker region, and a 65PRYH C-terminus with six disulfide bonds. In this dissertation, I used Rho as protein scaffold to study the interactions between integrins and disintegrins. Using cell adhesion assay, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and molecular docking, four structure-activity relationships between integrins and Rho mutants were identified: (1) the 48ARGDWN motif and C-terminus of Rho mutants acted synergistically and regulated the recognition of integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αVβ3 > α5β1. The 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant; however, the 48PRGDMP-65PRNPWNG mutant did not exhibit any integrin selectivity. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin αIIbβ3 indicated that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-pi interaction with the β3 K125 residue. Our results demonstrated that the RGD loop and C-terminus of disintegrins acted in a synergistic manner, resulting in their functional and structural differences in integrin binding; (2) Rho 48ARGDDP mutant selectively inhibited integrin αVβ3. To study the role of the C-terminal residue adjacent to the ARGD motif, we expressed Rho 48ARGD52XP mutants. The effect of the 52 residue position on their integrin binding activities was α5β1 (86-fold) > αIIbβ3 (41-fold) > αVβ3 (14-fold). The 48ARGDDP mutant was integrin αVβ3-specific mutant and inhibited integrins αVβ3, αIIbβ3, αVβ5, αVβ6, and α5β1 with the IC50 values of 45.3, 5117.2, 6886, 14980, and 5117.2 nM. X-ray structural analysis showed that the distance between the α carbons of Arg49 and Asp52 was 5.9 Å that is consistent with the structural requirement for integrin αVβ3-specific antagonist. The Met to Asp mutation caused a negative surface charge, which might be related to the lower activities toward integrins αIIbβ3, αVβ5, αVβ6, and α5β1. In vitro study showed that Rho 48ARGDDP mutant inhibited HUVEC migration and tube formation in a dose-dependent manner, suggesting its potential use as an anti-angiogenic agent; (3) the M to P mutation of the C-terminal residue adjacent to the ARGD motif abolished its binding to integrins. The 48ARGDPP mutant was an inactive integrin antagonist, which inhibited integrins αVβ3, α5β1, αIIbβ3, αVβ5, and αVβ6 with the IC50 values of 41260, 62460, 64665, 35247, and 15055.3 nM. X-ray structure analysis showed that Rho 48ARGDPP mutant has two conformations, and the P52 residue caused conformational change of the RGD motif. Met to Pro mutation in residue 52 caused the cis formation of D51-P52 peptide bond in conformer A, and that of P52-P53 in conformer B. The distance between the α carbons of Arg49 and Pro52 was increased up to 8.0 Å, indicating the disruption of turn conformation in the RGDX motif caused by the P52 residue. According to Ramachandran plot analysis, the P52 mutation modulated the D51 residue into an extended conformation and resulted in the loss of function of the RGDX motif. Our results demonstrated that the importance of the turn conformation in the RGDX motif of integrin ligands for integrin recognition; and (4) Rho 39KKARTICAR-48GRGDNP-65PRYH (KG) and 39KKARTICAR-48GRGDNP-65PGLYG (KG-P) mutants exhibited lower αIIbβ3 integrin inhibitory activity. The inhibitory activities of platelet aggregation by KG and KG-P mutants were 56 and 384 times lower than that by Rho. X-ray structural analyses of KG and KG-P mutants showed that their C-terminal regions interacted with the RGD loop: the R56 residue interacted with the Y67 residue in KG mutant, and the D55 residue interacted with the L67 and Y68 residues as well as the R49 and N52 residues interacted with Y68 residue in KG-P mutant. They had relatively narrower RGD loop and different electrostatic surface in comparison with those of Rho. The docking experiments showed that the positive charge patch formed by the R46 and R66 residues of Rho had salt bridge interactions with the negative charge D159 on the insert-3 region of αIIb subunit. KG and KG-P mutants did not have the positive charge patch due to the lack of the R46 residue in KG mutant, and the lack of the R46 and R66 residues in KG-P mutant. These results suggested that this positive charge patch may be important for the interaction of integrin αIIbβ3 with disintegrins. Our results demonstrated that the RGD loop, the linker region, and C-terminus of disintegrins acted in a synergistic manner, resulting in their functional and structural differences in integrin binding.

Akiyama, S.K. (2001). Purification of vitronectin. Curr Protoc Cell Biol Chapter 10, Unit 10 16.
Alghisi, G.C., Ponsonnet, L., and Ruegg, C. (2009). The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells. PLoS One 4, e4449.
Au, L.C., Huang, Y.B., Huang, T.F., Teh, G.W., Lin, H.H., and Choo, K.B. (1991). A common precursor for a putative hemorrhagic protein and rhodostomin, a platelet aggregation inhibitor of the venom of Calloselasma rhodostoma: molecular cloning and sequence analysis. Biochem Biophys Res Commun 181, 585-593.
Barczyk, M., Carracedo, S., and Gullberg, D. (2010). Integrins. Cell Tissue Res 339, 269-280.
Bartlett, G.J., Choudhary, A., Raines, R.T., and Woolfson, D.N. (2010). n-->pi* interactions in proteins. Nat Chem Biol 6, 615-620.
Benoit, Y.D., Groulx, J.F., Gagne, D., and Beaulieu, J.F. (2012). RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt. J Signal Transduct 2012, 248759.
Benvenuti, M., and Mangani, S. (2007). Crystallization of soluble proteins in vapor diffusion for x-ray crystallography. Nat Protoc 2, 1633-1651.
Brunger, A.T. (1992). X-PLOR :version 3.1 : a system for x-ray crystallography and NMR (New Haven: Yale University Press).
Byron, A., Humphries, J.D., Askari, J.A., Craig, S.E., Mould, A.P., and Humphries, M.J. (2009). Anti-integrin monoclonal antibodies. J Cell Sci 122, 4009-4011.
Byzova, T.V., Goldman, C.K., Pampori, N., Thomas, K.A., Bett, A., Shattil, S.J., and Plow, E.F. (2000). A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 6, 851-860.
Calvete, J.J., Marcinkiewicz, C., Monleon, D., Esteve, V., Celda, B., Juarez, P., and Sanz, L. (2005). Snake venom disintegrins: evolution of structure and function. Toxicon 45, 1063-1074.
Calvete, J.J., Moreno-Murciano, M.P., Theakston, R.D., Kisiel, D.G., and Marcinkiewicz, C. (2003). Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering. Biochem J 372, 725-734.
Chan, P.-T. (2012). Development of Integrins αvβx and α5β1-specific Antagonists Using Rhodostomin as a Scaffold. (Tainan, National Cheng Kung University).
Chen, C.Y. (2005). Use of Rhodostomin to Study the Integrin Recognition Sequences and to Establish a Method for Preparing Amino-Acid-Type Selective Isotope Labeling of Proteins. (Tainan, National Cheng Kung University).
Chen, Y.C. (2012). The role of the RGD motif and C-terminus of Echistatin and Rhodostomin in recognition of integrins. (Tainan, National Cheng Kung University).
Chen, Y.C., Cheng, C.H., Shiu, J.H., Chang, Y.T., Chang, Y.S., Huang, C.H., Lee, J.C., and Chuang, W.J. (2012). Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin. Toxicon 60, 1342-1348.
Cox, D., Brennan, M., and Moran, N. (2010). Integrins as therapeutic targets: lessons and opportunities. Nature reviews Drug discovery 9, 804-820.
de Vries, S.J., van Dijk, M., and Bonvin, A.M. (2010). The HADDOCK web server for data-driven biomolecular docking. Nat Protoc 5, 883-897.
Dennis, M.S., Carter, P., and Lazarus, R.A. (1993). Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. Proteins 15, 312-321.
Dennis, M.S., Henzel, W.J., Pitti, R.M., Lipari, M.T., Napier, M.A., Deisher, T.A., Bunting, S., and Lazarus, R.A. (1990). Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci U S A 87, 2471-2475.
Desgrosellier, J.S., and Cheresh, D.A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 10, 9-22.
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132.
Evans, R., Patzak, I., Svensson, L., De Filippo, K., Jones, K., McDowall, A., and Hogg, N. (2009). Integrins in immunity. J Cell Sci 122, 215-225.
Eyles, S.J., and Gierasch, L.M. (2000). Multiple roles of prolyl residues in structure and folding. J Mol Biol 301, 737-747.
Faber, K.N., Harder, W., Ab, G., and Veenhuis, M. (1995). Review: methylotrophic yeasts as factories for the production of foreign proteins. Yeast 11, 1331-1344.
Felding-Habermann, B., O'Toole, T.E., Smith, J.W., Fransvea, E., Ruggeri, Z.M., Ginsberg, M.H., Hughes, P.E., Pampori, N., Shattil, S.J., Saven, A., and Mueller, B.M. (2001). Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci U S A 98, 1853-1858.
Fujii, Y., Okuda, D., Fujimoto, Z., Horii, K., Morita, T., and Mizuno, H. (2003). Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD. J Mol Biol 332, 1115-1122.
Furie, B. (2009). Pathogenesis of thrombosis. Hematology Am Soc Hematol Educ Program, 255-258.
Gallivan, J.P., and Dougherty, D.A. (1999). Cation-pi interactions in structural biology. Proc Natl Acad Sci U S A 96, 9459-9464.
Goodman, S.L., and Picard, M. (2012). Integrins as therapeutic targets. Trends Pharmacol Sci 33, 405-412.
Gould, R.J., Polokoff, M.A., Friedman, P.A., Huang, T.F., Holt, J.C., Cook, J.J., and Niewiarowski, S. (1990). Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Biol Med 195, 168-171.
Grant, D.S., Tashiro, K., Segui-Real, B., Yamada, Y., Martin, G.R., and Kleinman, H.K. (1989). Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58, 933-943.
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. (2001). Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin. Proteins 43, 499-508.
Hinderaker, M.P., and Raines, R.T. (2003). An electronic effect on protein structure. Protein Sci 12, 1188-1194.
Honda, S., Kashiwagi, H., Kiyoi, T., Kato, H., Kosugi, S., Shiraga, M., Kurata, Y., and Tomiyama, Y. (2004). Amino acid mutagenesis within ligand-binding loops in alpha v confers loss-of-function or gain-of-function phenotype on integrin alpha v beta 3. Thromb Haemost 92, 1092-1098.
Horng, J.C., and Raines, R.T. (2006). Stereoelectronic effects on polyproline conformation. Protein Sci 15, 74-83.
Huang, T.F. (1998). What have snakes taught us about integrins? Cell Mol Life Sci 54, 527-540.
Huang, T.F., Holt, J.C., Lukasiewicz, H., and Niewiarowski, S. (1987a). Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. J Biol Chem 262, 16157-16163.
Huang, T.F., Sheu, J.R., Teng, C.M., Chen, S.W., and Liu, C.S. (1991). Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. J Biochem 109, 328-334.
Huang, T.F., Wu, Y.J., and Ouyang, C. (1987b). Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. Biochim Biophys Acta 925, 248-257.
Humphries, J.D., Byron, A., and Humphries, M.J. (2006). Integrin ligands at a glance. J Cell Sci 119, 3901-3903.
Hutchinson, E.G., and Thornton, J.M. (1994). A revised set of potentials for beta-turn formation in proteins. Protein Sci 3, 2207-2216.
Hynes, R.O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110, 673-687.
Juliano, D., Wang, Y., Marcinkiewicz, C., Rosenthal, L.A., Stewart, G.J., and Niewiarowski, S. (1996). Disintegrin interaction with alpha V beta 3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on beta 3 subunit. Exp Cell Res 225, 132-142.
Kay, L.E., Marion, D., and Bax, A. (1989). Practical Aspects of 3d Heteronuclear Nmr of Proteins. Journal of Magnetic Resonance 84, 72-84.
Kemperman, H., Wijnands, Y.M., and Roos, E. (1997). alphaV Integrins on HT-29 colon carcinoma cells: adhesion to fibronectin is mediated solely by small amounts of alphaVbeta6, and alphaVbeta5 is codistributed with actin fibers. Exp Cell Res 234, 156-164.
Khaspekova, S.G., Vyzova, T.V., Lukin, V.V., Tikhomirov, O., Berndt, M., Kouns, W., and Mazurov, A.V. (1996). [Conformational changes of the platelet membrane glycoprotein IIb-IIIa complex stimulated by a monoclonal antibody to the N-terminal segment of glycoprotein IIIa]. Biokhimiia 61, 412-428.
Koch, O., and Klebe, G. (2009). Turns revisited: a uniform and comprehensive classification of normal, open, and reverse turn families minimizing unassigned random chain portions. Proteins 74, 353-367.
Koradi, R., Billeter, M., and Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14, 51-55, 29-32.
Krammer, A., Lu, H., Isralewitz, B., Schulten, K., and Vogel, V. (1999). Forced unfolding of the fibronectin type III module reveals a tensile molecular recognition switch. Proc Natl Acad Sci U S A 96, 1351-1356.
Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. (1988). Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 107, 1589-1598.
Liang, T.-H. (2012). The Use of Rhodostomin to Study the Effect of C-terminal Region of Disintegrin on Recognition and Ligand-Induced Binding Site of Integrins. (Tainan, National Cheng Kung University).
Liau, C.-T. (2008). The Role of the Linker Region of Rhodostomin and Trimucin in Recognizing Integrins. (Tainan, National Cheng Kung University).
Liu, C.Z., Wang, Y.W., Shen, M.C., and Huang, T.F. (1994). Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry. Thromb Haemost 72, 919-925.
Locardi, E., Mullen, D.G., Mattern, R.H., and Goodman, M. (1999). Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3. J Pept Sci 5, 491-506.
Lovell, S.C., Davis, I.W., Arendall, W.B., 3rd, de Bakker, P.I., Word, J.M., Prisant, M.G., Richardson, J.S., and Richardson, D.C. (2003). Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50, 437-450.
Lu, X., Rahman, S., Kakkar, V.V., and Authi, K.S. (1996). 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.
Luft, J.R., Wolfley, J.R., and Snell, E.H. (2011). What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments. Cryst Growth Des 11, 651-663.
MacArthur, M.W., and Thornton, J.M. (1991). Influence of proline residues on protein conformation. J Mol Biol 218, 397-412.
Macauley-Patrick, S., Fazenda, M.L., McNeil, B., and Harvey, L.M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249-270.
Marcinkiewicz, C. (2005). Functional characteristic of snake venom disintegrins: potential therapeutic implication. Curr Pharm Des 11, 815-827.
Marcinkiewicz, C., Vijay-Kumar, S., McLane, M.A., and Niewiarowski, S. (1997). Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site. Blood 90, 1565-1575.
Mas-Moruno, C., Rechenmacher, F., and Kessler, H. (2010). Cilengitide: the first anti-angiogenic small molecule drug candidate design, synthesis and clinical evaluation. Anticancer Agents Med Chem 10, 753-768.
McCabe, N.P., De, S., Vasanji, A., Brainard, J., and Byzova, T.V. (2007). Prostate cancer specific integrin alphavbeta3 modulates bone metastatic growth and tissue remodeling. Oncogene 26, 6238-6243.
McDowall, A., Inwald, D., Leitinger, B., Jones, A., Liesner, R., Klein, N., and Hogg, N. (2003). A novel form of integrin dysfunction involving beta1, beta2, and beta3 integrins. J Clin Invest 111, 51-60.
McLane, M.A., Kowalska, M.A., Silver, L., Shattil, S.J., and Niewiarowski, S. (1994). Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets. Biochem J 301 ( Pt 2), 429-436.
McLane, M.A., Kuchar, M.A., Brando, C., Santoli, D., Paquette-Straub, C.A., and Miele, M.E. (2001). New insights on disintegrin-receptor interactions: eristostatin and melanoma cells. Haemostasis 31, 177-182.
McLane, M.A., Sanchez, E.E., Wong, A., Paquette-Straub, C., and Perez, J.C. (2004). Disintegrins. Curr Drug Targets Cardiovasc Haematol Disord 4, 327-355.
McLane, M.A., Vijay-Kumar, S., Marcinkiewicz, C., Calvete, J.J., and Niewiarowski, S. (1996). Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins. FEBS Lett 391, 139-143.
McLane, M.A., Zhang, X., Tian, J., Zelinskas, C., Srivastava, A., Hensley, B., and Paquette-Straub, C. (2005). Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells. Pathophysiol Haemost Thromb 34, 164-168.
Monleon, D., Esteve, V., Kovacs, H., Calvete, J.J., and Celda, B. (2005). Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR. Biochem J 387, 57-66.
Mousa, S.A. (1999). Antiplatelet therapies: from aspirin to GPIIb/IIIa-receptor antagonists and beyond. Drug Discov Today 4, 552-561.
Mullen, D.G., Cheng, S., Ahmed, S., Blevitt,, J.M., B., D., Craig, W.S., Ingram, R.T.,, Mazur, C., Minasyan, R., Tolley, J.O.,, and Tschopp, J.F., Pierschbacher, M.D. (1996). Development of peptide antagonists of the integrin αVβ3. Peptides: chemistry, structure and biology Proceedings of the 14th American Peptide Symposium, 207-208.
Murphy, M.G., Cerchio, K., Stoch, S.A., Gottesdiener, K., Wu, M., and Recker, R. (2005). Effect of L-000845704, an alphaVbeta3 integrin antagonist, on markers of bone turnover and bone mineral density in postmenopausal osteoporotic women. J Clin Endocrinol Metab 90, 2022-2028.
Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A.A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67, 355-367.
Nagae, M., Re, S., Mihara, E., Nogi, T., Sugita, Y., and Takagi, J. (2012). Crystal structure of alpha5beta1 integrin ectodomain: atomic details of the fibronectin receptor. J Cell Biol 197, 131-140.
Nilges, M., Clore, G.M., and Gronenborn, A.M. (1988). Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding. FEBS Lett 239, 129-136.
Niu, G., and Chen, X. (2011). Why integrin as a primary target for imaging and therapy. Theranostics 1, 30-47.
Niu, J., Gu, X., Ahmed, N., Andrews, S., Turton, J., Bates, R., and Agrez, M. (2001). The alphaVbeta6 integrin regulates its own expression with cell crowding: implications for tumour progression. Int J Cancer 92, 40-48.
Page, R., and Stevens, R.C. (2004). Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens. Methods 34, 373-389.
Park, H.S., Kim, C., and Kang, Y.K. (2002). Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets. Biopolymers 63, 298-313.
Patel, D.H., Wi, S.G., and Bae, H.J. (2009). Modification of overlap extension PCR: A mutagenic approach. Indian J Biotechnol 8, 183-186.
Peishoff, C.E., Ali, F.E., Bean, J.W., Calvo, R., D'Ambrosio, C.A., Eggleston, D.S., Hwang, S.M., Kline, T.P., Koster, P.F., Nichols, A., and et al. (1992). Investigation of conformational specificity at GPIIb/IIIa: evaluation of conformationally constrained RGD peptides. J Med Chem 35, 3962-3969.
Peter, K., Schwarz, M., Nordt, T., and Bode, C. (2001). Intrinsic activating properties of GP IIb/IIIa blockers. Thromb Res 103 Suppl 1, S21-27.
Potterton, E., Briggs, P., Turkenburg, M., and Dodson, E. (2003). A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59, 1131-1137.
Rahman, S., Aitken, A., Flynn, G., Formstone, C., and Savidge, G.F. (1998). 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 ( Pt 2), 247-257.
Regnault, V., Rivat, C., Maugras, M., and Stoltz, J.F. (1988a). Highly purified, functionally active human fibronectin preparation. Rev Fr Transfus Immunohematol 31, 19-34.
Regnault, V., Rivat, C., and Stoltz, J.F. (1988b). Affinity purification of human plasma fibronectin on immobilized gelatin. J Chromatogr 432, 93-102.
Reimer, U., Scherer, G., Drewello, M., Kruber, S., Schutkowski, M., and Fischer, G. (1998). Side-chain effects on peptidyl-prolyl cis/trans isomerisation. J Mol Biol 279, 449-460.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12, 697-715.
Saha, I., and Shamala, N. (2012). Investigating diproline segments in proteins: occurrences, conformation and classification. Biopolymers 97, 54-64.
Sameh Sarray, J.L., Mohamed El Ayeb and Naziha Marrakchi (2013). Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects. Bioactive Food Peptides in Health and Disease, 219-238.
Scarborough, R.M., Naughton, M.A., Teng, W., Rose, J.W., Phillips, D.R., Nannizzi, L., Arfsten, A., Campbell, A.M., and Charo, I.F. (1993a). Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J Biol Chem 268, 1066-1073.
Scarborough, R.M., Rose, J.W., Naughton, M.A., Phillips, D.R., Nannizzi, L., Arfsten, A., Campbell, A.M., and Charo, I.F. (1993b). Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem 268, 1058-1065.
Selistre-de-Araujo, H.S., Pontes, C.L., Montenegro, C.F., and Martin, A.C. (2010). Snake venom disintegrins and cell migration. Toxins 2, 2606-2621.
Sem, D.S., and Pellecchia, M. (2001). NMR in the acceleration of drug discovery. Curr Opin Drug Discov Devel 4, 479-492.
Senn, H., and Klaus, W. (1993). The nuclear magnetic resonance solution structure of flavoridin, an antagonist of the platelet GP IIb-IIIa receptor. J Mol Biol 232, 907-925.
Shiu, J.-H. (2012). Structure, Dynamics, and Function Relationships of Rhodostomin Mutants and Variants: Insight into their Interactions with Integrins. (Tainan, National Cheng Kung University).
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. (2004). Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding. Proteins 57, 839-849.
Shiu, J.H., Chen, C.Y., Chen, Y.C., Chang, Y.T., Chang, Y.S., Huang, C.H., and Chuang, W.J. (2012). 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.
Springer, T.A., and Wang, J.H. (2004). The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Adv Protein Chem 68, 29-63.
Sumathipala, R., Xu, C., Seago, J., Mould, A.P., Humphries, M.J., Craig, S.E., Patel, Y., Wijelath, E.S., Sobel, M., and Rahman, S. (2006). The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity. J Biol Chem 281, 37686-37696.
Takada, Y., Ye, X., and Simon, S. (2007). The integrins. Genome Biol 8, 215.
Takagi, J., Kamata, T., Meredith, J., Puzon-McLaughlin, W., and Takada, Y. (1997). Changing ligand specificities of alphavbeta1 and alphavbeta3 integrins by swapping a short diverse sequence of the beta subunit. J Biol Chem 272, 19794-19800.
Taylor, G.L. (2010). Introduction to phasing. Acta Crystallogr D Biol Crystallogr 66, 325-338.
Tofteng, C.L., Bach-Mortensen, P., Bojesen, S.E., Tybjaerg-Hansen, A., Hyldstrup, L., and Nordestgaard, B.G. (2007). Integrin beta3 Leu33Pro polymorphism and risk of hip fracture: 25 years follow-up of 9233 adults from the general population. Pharmacogenet Genomics 17, 85-91.
Torshin, I.Y. (2002). Structural criteria of biologically active RGD-sites for analysis of protein cellular function - a bioinformatics study. Med Sci Monit 8, BR301-312.
Vaguine, A.A., Richelle, J., and Wodak, S.J. (1999). SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr D Biol Crystallogr 55, 191-205.
Van Agthoven, J.F., Xiong, J.P., Alonso, J.L., Rui, X., Adair, B.D., Goodman, S.L., and Arnaout, M.A. (2014). Structural basis for pure antagonism of integrin alphaVbeta3 by a high-affinity form of fibronectin. Nat Struct Mol Biol 21, 383-388.
Wagner, G., Braun, W., Havel, T.F., Schaumann, T., Go, N., and Wuthrich, K. (1987). Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN. J Mol Biol 196, 611-639.
Wedemeyer, W.J., Welker, E., and Scheraga, H.A. (2002). Proline cis-trans isomerization and protein folding. Biochemistry 41, 14637-14644.
Wierzbicka-Patynowski, I., Niewiarowski, S., Marcinkiewicz, C., Calvete, J.J., Marcinkiewicz, M.M., and McLane, M.A. (1999). Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins. J Biol Chem 274, 37809-37814.
Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G., McCoy, A., McNicholas, S.J., Murshudov, G.N., Pannu, N.S., Potterton, E.A., Powell, H.R., Read, R.J., Vagin, A., and Wilson, K.S. (2011). Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235-242.
Wishart, D. (2005). NMR spectroscopy and protein structure determination: applications to drug discovery and development. Curr Pharm Biotechnol 6, 105-120.
Wuthrich, K. (1986). NMR of proteins and nucleic acids (New York: John Wiley & Sons).
Xiao, T., Takagi, J., Coller, B.S., Wang, J.H., and Springer, T.A. (2004). Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432, 59-67.
Xiong, J.P., Mahalingham, B., Alonso, J.L., Borrelli, L.A., Rui, X., Anand, S., Hyman, B.T., Rysiok, T., Muller-Pompalla, D., Goodman, S.L., and Arnaout, M.A. (2009). Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment. J Cell Biol 186, 589-600.
Xiong, J.P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D.L., Joachimiak, A., Goodman, S.L., Arnaout, M.A.C.I.N.S.O., and Pmid (2001). Crystal structure of the extracellular segment of integrin alphaVbeta3. Science 294, 339-345.
Xiong, J.P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., and Arnaout, M.A. (2002). Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science 296, 151-155.
Yeh, C.H., Peng, H.C., Yang, R.S., and Huang, T.F. (2001). Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells. Mol Pharmacol 59, 1333-1342.
Zbyszek Otwinowski, W.M. (1997). Processing of X-ray diffraction data collected in oscillation mode. In Methods in Enzymology, Charles W. Carter, Jr., ed., pp. 307-326.
Zhu, J., Zhu, J., and Springer, T.A. (2013). Complete integrin headpiece opening in eight steps. J Cell Biol 201, 1053-1068.