The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.
The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.
Martin
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PD
Department of Biochemistry, Wayne State University, Detroit, Michigan 48201.
Robertson
W
W
Turk
D
D
Huber
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R
Bode
W
W
Edwards
B F
BF
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013917:Thrombin / Q000378:metabolism*; D014961:X-Ray Diffraction
D000590:Amino Acid Chloromethyl Ketones; D010446:Peptide Fragments; C038145:prolyl-phenylalanyl-arginine chloromethyl ketone; D005344:Fibrinopeptide A; D005340:Fibrinogen; D013917:Thrombin