Concerted Motions of the Integrin-binding Loop and the C-terminal Tail of the Non-RGD Disintegrin Obtustatin

Abstract

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...

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

“…The KTD motif of NtACP is part of the G-I loop that structural and topological alignment procedures have identified as analogous to the loop containing the RGD motif of FnIII10 (35,40). Although integrins generally bind RGD sequences, several proteins have been shown to bind a number of related motifs, such as KTS (42,43), MLD (44), or MGD(W) (45) of disintegrins obtustatin, EC3, and EMF-10, respectively. Similarly, COL15, the largest collagenous domain of type XVII collagen, binds to the ␣ 5 ␤ 1 and ␣ V ␤ 1 integrins through a KGD motif (46).…”

Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis

“…It is worth noting that the KTS loop and the C-terminal domain are in close spatial proximity and located on the same side of the molecule. 40 It is therefore likely that both Work is in progress in our laboratory to synthesize lebestatin analogues in order to better understand structure-activity relationships of these proteins and to define the minimum structures of a1b1 receptor.…”

NMRstructure of bitistatin – a missing piece in the evolutionary pathway of snake venom disintegrins

“…Apart from the sequence of the integrin-recognition tripeptide motif, the dynamics of the binding loop is hypothesized to play a role modulating the binding affinity of the disintegrin towards its target integrin receptor [42,53]. In the case of bitistatin, residues Arg64-Gly65-Asp66 are located between the hydrophobic side-chains of Ile62 (strictly conserved in disintegrins) and Trp67 (Phe in flavoridin and trimestatin; Met in kistrin).…”

“…In addition, a number of NMR structures of medium-sized (kistrin, 1KST [53]; flavoridin, 1FVL [38]; albolabrin [39]; salmosin, 1L3X [40]; rhodostomin, 1Q7J, 1Q7I, 2PJI, 2PJF, 2PJG, 2LJV, 3UCI, 2M75, 2M7F, 2M7H) and short (echistatin, 2ECH [41]; 1RO3 [42]; obtustatin, 1MPZ [43]; jerdostatin, 2W9O, 2W9U, 2W9V, 2W9W [44]) disintegrins, and the crystal structures of the medium-sized disintegrin trimestatin (1J2L) [45] and dimeric disintegrins (schistatin, 1Z1X, 1RMR, 1TEJ [23,24]; acostatin (3CO5) [46]), have been reported. The crystal structure of the disintegrin-like and cysteine-rich domains of ADAM-10 has also been solved (2AO7) [47].…”