3-DIMENSIONAL STRUCTURE OF ECHISTATIN AND DYNAMICS OF THE ACTIVE-SITE

The snake venom protein echistatin contains the cell recognition seque nce Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the act ive RGD site are presented. A set of structures was determined using t he Distance Geometry method and subsequently refined by Molecular Dyna mics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser eff ect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment fro m Gly(5) to Cys(20) and from Asp(30) to Asn(42) has a well-defined con formation and the Arg-Gly-Asp sequence, which adopts a turn-like struc ture, is located at the apex of a nine-residue loop connecting the two strands of a distorted P-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by N-15 relaxation measurements. The overall correlation time of echistatin was determined from fluore scence measurements, and was used in a model-free analysis to determin e internal motional parameters. The active site has order parameters o f 0.3-0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution struct ures was made by calculating generalized order parameters from the ens emble of three-dimensional structures. The motion of the RGD site dete cted experimentally is more extensive than a simple RGD loop 'wagging' motional model, suggested by an examination of superposed solution st ructures.