We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin alpha5beta1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the alpha4beta1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both alpha5beta1 and alpha4beta1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (a disintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.
Obtustatin is a potent and selective inhibitor of the ␣ 1  1 integrin in vitro and of angiogenesis in vivo. It possesses an integrin recognition loop that harbors, in a lateral position, the inhibitory 21 KTS 23 motif. We report an analysis of the dynamics of the backbone and sidechain atoms of obtustatin by homonuclear NMR methods. Angular mobility has been calculated for 90 assigned cross-peaks from 22 off-resonance rotating frame nuclear Overhauser effect spectroscopy spectra recorded at three magnetic fields. Our results suggest that the integrin binding loop and the C-terminal tail display concerted motions, which can be interpreted by hinge effects. Among the integrin-binding motif, threonine 22 and serine 23 exhibit the lowest and the highest sidechain flexibility, respectively. It is noteworthy that the side chain of threonine 22 is not solvent-exposed, although based on synthetic peptides it appears to be the most critical residue for the inhibitory activity of obtustatin on the binding of integrin ␣ 1  1 to collagen IV. Instead, the side chain of threonine 22 is oriented toward the loop center and hydrogen-bonded to residues Thr 25 and Ser 26 . This network of interactions explains the restrained mobility of threonine 22 and suggests that its functional importance lies in maintaining the active conformation of the ␣ 1  1 inhibitory loop.Disintegrins represent a group of cysteine-rich peptides released in Crotalidae and Viperidae snake venoms by proteolytic processing of PII metalloproteinases (1). Disintegrins are potent inhibitors of the binding of  1 and  3 integrins to their ligands. Biochemical (2,3) and structural studies (4 -7) have disclosed that the inhibitory activity of disintegrins is linked to a tripeptide motif, which mimics the recognition sequence of the integrin ligands. The tripeptide is maintained in the active conformation at the apex of a mobile loop by the appropriate pairing of cysteine residues (8, 9).Disintegrins are divided into five different groups (10). The first group includes short disintegrins composed of 49 -51 residues and four disulfide bonds. The second group is formed by medium sized disintegrins, which contain about 70 amino acids and six disulfide bonds. The third group includes long disintegrins composed of 84 residues cross-linked by seven disulfide bonds. The disintegrin domains of PIII snake venom metalloproteases contain about 100 amino acids with 16 cysteine residues involved in the formation of eight disulfide bonds. These constitute the fourth group of the disintegrin family. Unlike the short, medium, and long disintegrins, which are single-chain molecules, the fifth group is composed of homo-and heterodimers. Dimeric disintegrins contain subunits of about 67 residues with 10 cysteines involved in the formation of four intrachain disulfide bonds and two interchain cysteine linkages. Current biochemical and genetic data support the view that structural diversification among the disintegrin family occurred through disulfide bond engineering (10).The inte...
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