Peptide name : Disintegrin metalloproteinase/disintegrin echistatin

Source/Organism : Saw-scaled viper

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 186

C-terminal modification: Not found

N-terminal modification : Free

Non-natural peptide information: None

Assay type : Transwell migration assay

Assay time : 6h

Activity : IC50 : 154.5 μM

Cell line : Panc-1

Cancer type : MG human glioblastoma

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 20363.8242 Dalton

Aliphatic index : 0.529

Instability index : 31.1882

Hydrophobicity (GRAVY) : -0.644

Isoelectric point : 6.1603

Charge (pH 7) : -2.7646

Aromaticity : 0.048

Molar extinction coefficient (cysteine, cystine): (10430, 11430)

Hydrophobic/hydrophilic ratio : 0.89795918

hydrophobic moment : -0.07

Missing amino acid : W

Most occurring amino acid : G

Most occurring amino acid frequency : 17

Least occurring amino acid : F

Least occurring amino acid frequency : 2

Secondary structure fraction (Helix, Turn, Sheet): (0.2, 0.3, 0.2)

SMILES Notation: CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)NCC(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)NCC(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)O)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)CC)C(C)C)C(C)C)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)CC

1 : Monleón D, et al. Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR. Biochem J. 2005; 387:57-66. doi: 10.1042/BJ20041343

2 : Casewell NR, et al. Gene tree parsimony of multilocus snake venom protein families reveals species tree conflict as a result of multiple parallel gene loss. Mol Biol Evol. 2011; 28:1157-72. doi: 10.1093/molbev/msq302

3 : Cooke RM, et al. Nuclear magnetic resonance studies of the snake toxin echistatin. 1H resonance assignments and secondary structure. Eur J Biochem. 1991; 202:323-8. doi: 10.1111/j.1432-1033.1991.tb16379.x

4 : Calvete JJ, et al. The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin. FEBS Lett. 1992; 309:316-20. doi: 10.1016/0014-5793(92)80797-k

5 : Atkinson RA, et al. Echistatin: the refined structure of a disintegrin in solution by 1H NMR and restrained molecular dynamics. Int J Pept Protein Res. 1994; 43:563-72. doi: 10.1111/j.1399-3011.1994.tb00558.x

6 : Saudek V, et al. Three-dimensional structure of echistatin, the smallest active RGD protein. Biochemistry. 1991; 30:7369-72. doi: 10.1021/bi00244a003

7 : Dalvit C, et al. 1H NMR studies of echistatin in solution. Sequential resonance assignments and secondary structure. Eur J Biochem. 1991; 202:315-21. doi: 10.1111/j.1432-1033.1991.tb16378.x

8 : Dennis MS, et al. Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci U S A. 1990; 87:2471-5. doi: 10.1073/pnas.87.7.2471

9 : Carson F, et al. Examining Negative Emotional Symptoms and Psychological Wellbeing of Australian Sport Officials. Int J Environ Res Public Health. 2020; 17:(unknown pages). doi: 10.3390/ijerph17218265

10 : Topol EJ, et al. Platelet GPIIb-IIIa blockers. Lancet. 1999; 353:227-31. doi: 10.1016/S0140-6736(98)11086-3

11 : Chen Y, et al. Proton NMR assignments and secondary structure of the snake venom protein echistatin. Biochemistry. 1991; 30:11625-36. doi: 10.1021/bi00114a004

12 : Marcinkiewicz C, et al. 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. 1997; 90:1565-75.

13 : Saudek V, et al. The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy. Eur J Biochem. 1991; 202:329-38. doi: 10.1111/j.1432-1033.1991.tb16380.x

14 : Gan ZR, et al. Echistatin. A potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus. J Biol Chem. 1988; 263:19827-32.

Paper title : Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Doi : https://doi.org/10.1042/BJ20041343

Abstract : Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Paper title : Gene tree parsimony of multilocus snake venom protein families reveals species tree conflict as a result of multiple parallel gene loss.

Doi : https://doi.org/10.1093/molbev/msq302

Abstract : The proliferation of gene data from multiple loci of large multigene families has been greatly facilitated by considerable recent advances in sequence generation. The evolution of such gene families, which often undergo complex histories and different rates of change, combined with increases in sequence data, pose complex problems for traditional phylogenetic analyses, and in particular, those that aim to successfully recover species relationships from gene trees. Here, we implement gene tree parsimony analyses on multicopy gene family data sets of snake venom proteins for two separate groups of taxa, incorporating Bayesian posterior distributions as a rigorous strategy to account for the uncertainty present in gene trees. Gene tree parsimony largely failed to infer species trees congruent with each other or with species phylogenies derived from mitochondrial and single-copy nuclear sequences. Analysis of four toxin gene families from a large expressed sequence tag data set from the viper genus Echis failed to produce a consistent topology, and reanalysis of a previously published gene tree parsimony data set, from the family Elapidae, suggested that species tree topologies were predominantly unsupported. We suggest that gene tree parsimony failure in the family Elapidae is likely the result of unequal and/or incomplete sampling of paralogous genes and demonstrate that multiple parallel gene losses are likely responsible for the significant species tree conflict observed in the genus Echis. These results highlight the potential for gene tree parsimony analyses to be undermined by rapidly evolving multilocus gene families under strong natural selection.

Paper title : Nuclear magnetic resonance studies of the snake toxin echistatin. 1H resonance assignments and secondary structure.

Doi : https://doi.org/10.1111/j.1432-1033.1991.tb16379.x

Abstract : The 1H-NMR spectrum of the snake toxin echistatin has been assigned using homonuclear two-dimensional methods. Consideration of the NOE patterns, coupling constants and putative hydrogen bonds enabled two regular features of secondary structure to be deduced: a beta-sheet/turn between residues 8 and 13 and a small anti-parallel beta-sheet and bulge linking residues 16-20 with residues 30-33. The recognition region of the protein containing the residues RGD lies in a loop joining the two strands of the beta-sheet. The beta-bulge and the loop containing the RGD sequence undergo pH-dependent conformational interconversion, modulated by the side chain of Asp29.

Paper title : The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Doi : https://doi.org/10.1016/0014-5793(92)80797-k

Abstract : Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.

Paper title : Echistatin: the refined structure of a disintegrin in solution by 1H NMR and restrained molecular dynamics.

Doi : https://doi.org/10.1111/j.1399-3011.1994.tb00558.x

Abstract : The structure of the disintegrin echistatin has been determined by 1H NMR, distance geometry calculations and restrained molecular dynamics simulations. The structure has been refined from the preliminary distance geometry calculations with the inclusion of additional 1H NMR data and hydrogen bonds identified in early stages of the molecular dynamics calculations. The calculations reported here allow a distinction to be made between the two possible disulfide bridging patterns-echistatin is crosslinked as follows: Cys2-Cys11, Cys7-Cys32, Cys8-Cys37, Cys20-Cys39. The final set of structures gives an average pairwise root mean square distance of 0.100 nm (calculated over the backbone atoms of residues Ser4-Cys20 and Asp30-Pro40). The core of echistatin is a well defined though irregular structure, composed of a series of non-classical turns crosslinked by the disulfide bridges and stabilised by hydrogen bonds. The RGD sequence is located in a protruding loop whose stem is formed by two rigid, hydrogen-bonded strands (Thr18-Cys20, Asp30-Cys32). The RGD sequence is connected to this structure by short, flexible segments. High (but not unlimited) mobility is probably necessary for fast recognition and fitting to the integrin receptors. Sequence variability among the disintegrins is found in the segments flanking the RGD sequence, suggesting that these may be important in conferring specificity for the receptors.

Paper title : Three-dimensional structure of echistatin, the smallest active RGD protein.

Doi : https://doi.org/10.1021/bi00244a003

Abstract : Echistatin is a 49 amino acid protein isolated from the venom of a viper (Echis carinatus) and is one of the smallest natural adhesive ligands that interacts with integrin-type receptors through an Arg-Gly-Asp (RGD) sequence. The structure of echistatin in aqueous solution has been determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 490 interatomic distance constraints, which were used in distance geometry calculations. The chain is shown to fold in a series of irregular loops to form a rigid core stabilized by four cystine cross-links. From this core an irregular hairpin and the C-terminus protrude. The core and the hairpin are further stabilized by a network of hydrogen bonds. The RGD sequence is located in a mobile loop at the tip of the hairpin. The mobility and its significance for activity are discussed.

Paper title : 1H NMR studies of echistatin in solution. Sequential resonance assignments and secondary structure.

Doi : https://doi.org/10.1111/j.1432-1033.1991.tb16378.x

Abstract : Two-dimensional 1H-NMR methods have been used to obtain complete proton resonance assignments for the 49-residue protein echistatin from the viper Echis carinatus. The protein in solution contains only a small amount of regular secondary structure with four very short beta-strands. These beta-strands form two short segments of antiparallel beta-sheet, as evidenced by the observed cross-strand NOE. The first two strands are connected with a tight reverse turn, whereas the remaining two strands are linked together by an 11-residue loop forming a so-called hairpin. The tripeptide unit Arg-Gly-Asp, responsible for the binding of echistatin to the fibrinogen receptor glycoprotein GPIIb/IIIa, is located at the tip of this very hydrophilic loop.

Paper title : Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors.

Doi : https://doi.org/10.1073/pnas.87.7.2471

Abstract : The purification, complete amino acid sequence, and biological activity are described for several homologous snake venom proteins that are platelet glycoprotein (GP) IIb-IIIa antagonists and potent inhibitors of platelet aggregation. The primary structures of kistrin (from Agkistrodon rhodostoma), bitan (from Bitis arietans), three isoforms of trigramin (from Trimeresusus gramineus), and an isoform of echistatin (from Echis carinatus) were determined by automated sequence analysis and fast atom bombardment mass spectrometry analysis. Each of the protein in this family, which range from 47 to 83 residues, contains an Arg-Gly-Asp amino acid sequence found in protein ligands that binds to GPIIb-IIIa, a high (17 +/- 1%) cysteine content conserved in the primary sequence, and a homologous N-terminal region absent only in the echistatin isoforms. Each protein directly inhibits the interaction of purified platelet GPIIb-IIIa to immobilized fibrinogen about 100 times more effectively than does the pentapeptide Gly-Arg-Gly-Asp-Ser; IC50 values range from 1.1 to 3.0 nM. The IC50 value for the inhibition of platelet aggregation, using human platelet-rich plasma stimulated with ADP, ranges from 110 to 550 nM for the various proteins, about 1000-fold more potent than Gly-Arg-Gly-Asp-Ser. Kistrin binds reversibly to both resting and ADP-activated human platelets with high affinity (Kd = 10.8 nM and 1.7 nM, respectively) and to purified GPIIb-IIIa with a lower affinity (Kd = approximately 100 nM). Finally, kistrin injected at 1.0 mg/kg into rabbits reversibly inhibits platelet aggregation ex vivo over 30 min without induction of thrombocytopenia. We propose that these proteins are members of a general class of proteins found in the venom of pit vipers that inhibit platelet aggregation by antagonism of the GPIIb-IIIa-fibrinogen interaction and as such serve as potential antithrombotic agents.

Paper title : Examining Negative Emotional Symptoms and Psychological Wellbeing of Australian Sport Officials.

Doi : https://doi.org/10.3390/ijerph17218265

Abstract : Sports officials are exposed to numerous performance and personal stressors, however little is known about their mental health and psychological wellbeing. This study investigated levels of mental health and psychological wellbeing of sports officials in Australia, and the demographic, officiating, and workplace factors associated with these outcomes. An online survey consisting of demographic and officiating questions, and measures of work engagement, mental health and psychological wellbeing was completed by 317 officials. A negative emotional symptoms score was computed. Associations between key demographic, officiating, and workplace factors with negative emotional symptoms and psychological wellbeing were assessed using univariate and multivariate analyses. Officials who were younger, not in a committed relationship, having lower levels of education, and less officiating experience reported higher levels of negative emotional symptoms, while males, older than 50 years, in a committed relationship and more officiating experience had higher levels of psychological wellbeing. The ability to self-manage workload and demonstrate professional autonomy were strongly associated with negative emotional symptoms and psychological wellbeing. Officials reported high negative emotional symptoms, but also high levels of psychological wellbeing. The ability to manage workload and to express professional autonomy are important determinants of mental health and wellbeing levels of sports officials.

Paper title : Platelet GPIIb-IIIa blockers.

Doi : https://doi.org/10.1016/S0140-6736(98)11086-3

Abstract : Regardless of the event that stimulates the aggregation of platelets, the receptor alpha(IIb)beta3--one of a family of adhesion receptors known as integrins--has a key role in the process. The past decade has seen the publication of 10 phase III (randomised) clinical trials of four members of a new class of antiplatelet drugs, the GPIIb-IIIa blockers, targeted at this important receptor. Three (abciximab, eptifibatide, and tirofiban) are licensed for human use. 10 other GbIIb-IIIa blockers are in phase II or III human studies. In all 10 placebo-controlled trials, done in the clinical settings of percutaneous coronary intervention or acute coronary syndrome in patients on aspirin, the endpoints favoured the active drug, with a risk reduction for death or non-fatal myocardial infarction of about 21% overall. With attention to heparin dose the risk of bleeding is not a major concern with these agents. The GPIIb-IIIa blockers are taking the clinician and patient out of the era of aspirin monotherapy when platelet inhibition is required.

Paper title : Proton NMR assignments and secondary structure of the snake venom protein echistatin.

Doi : https://doi.org/10.1021/bi00114a004

Abstract : The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.

Paper title : 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.

Doi : https://doi.org/Not available

Abstract : Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to alpha(v) beta3 and alphaIIb beta3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with alphaIIb beta3 and alpha(v) beta3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to alphaIIb beta3 and decreased binding to alpha(v) beta3. This substitution impaired the ability of echistatin to induce LIBS in alpha(v) beta3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind alphaIIb beta3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with alphaIIb beta3 and alpha(v) beta3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized alpha(v) beta3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified alpha(v) beta3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of alphaIIb beta3 and alpha(v) beta3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

Paper title : The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Doi : https://doi.org/10.1111/j.1432-1033.1991.tb16380.x

Abstract : Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.

Paper title : Echistatin. A potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus.

Doi : https://doi.org/Not available

Abstract : A 49-residue protein, echistatin, which inhibits platelet aggregation, was purified from the venom of the saw-scaled viper Echis carinatus. The purification procedure included gel filtration on Sephadex G-50, cation-exchange chromatography on Mono S, and C18 reverse-phase high pressure liquid chromatography. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, reverse-phase high pressure liquid chromatography, and NH2-terminal sequence analysis. Echistatin is a single-chain polypeptide with a molecular weight of 5400 and a native isoelectric point of 8.3. The most abundant amino acid, cysteine, accounts for 8 of the 49 residues in the protein. A 10-residue segment of echistatin shows 90% identity to a portion of the sequence of trigramin, a platelet aggregation inhibitor from the green tree viper Trimereserus gramineus (Huang, T.-F., Holt, J. C., Lukasiewicz, H., and Niewiarowski, S. (1987) J. Biol. Chem. 262, 16157-16163). Echistatin contains the sequence arginine-glycine-aspartic acid, which is common to proteins which bind to the glycoprotein IIb/IIIa complex. It also contains the sequence proline-arginine-asparagine-proline, which is found in the A alpha chain of human fibrinogen at position 267-270. The purified protein inhibits fibrinogen-dependent platelet aggregation initiated by ADP with an IC50 of 3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of echistatin abolished its inhibitory activity.