Recombinant decorsin: dynamics of the RGD recognition site.
Decorsin is an antagonist of integrin alphaIIbbeta3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate b-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 the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities.
Krezel
A M
AM
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA. Andrzej.M.Krezel@vanderbilt.edu
Ulmer
J S
JS
Wagner
G
G
Lazarus
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015815:Cell Adhesion Molecules; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007865:Leeches / Q000737:chemistry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D015815:Cell Adhesion Molecules; D017931:DNA Primers; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011994:Recombinant Proteins; C064478:decorsin; C047981:arginyl-glycyl-aspartic acid