Recombinant decorsin: Dynamics of the RGD recognition site

Decorsin is an antagonist of integrin alpha(IIb)beta(3) and a potent platel et aggregation inhibitor. A synthetic gene encoding decorsin, originally is olated from the leech Macrobdella decora, was designed, constructed, and ex pressed in Escherichia coli. The synthetic gene was fused to the stII signa l sequence and expressed under the transcriptional control of the E. coli a lkaline phosphatase promoter. The protein was purified by size-exclusion fi ltration of the periplasmic contents followed by reversed-phase high-perfor mance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid compositi on, mass spectral analysis, and biological activity assays. Complete sequen tial assignments of H-1 and proton bound C-13 resonances were established. Stereospecific assignments of 21 of 25 nondegenerate beta-methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although th e loop containing the RGD sequence is the most flexible one in decorsin, th e conformation of the RGD site itself is more restricted than in other prot eins with similar activities.