CRYSTAL-STRUCTURE OF FIBRINOGEN-A-ALPHA PEPTIDE-1-23 (F8Y) BOUND TO BOVINE THROMBIN EXPLAINS WHY THE MUTATION OF PHE-8 TO TYROSINE STRONGLYINHIBITS NORMAL CLEAVAGE AT ARG-16

A peptide containing residues 1-50 of the A alpha-chain of fibrinogen, expressed as a fusion peptide with beta-galactosidase, is rapidly cle aved by thrombin at Arg-16, similarly to whole fibrinogen. When Phe-g, which is highly conserved, is replaced with tyrosine (F8Y), the cleav age is slowed drastically [Lord, Byrd, Hede, Wei and Colby (1990) J. B iol. Chem. 265, 838-843]. To examine the structural basis for this res ult, we have determined the crystal structure of bovine thrombin compl exed with a synthetic peptide containing residues 1-23 of fibrinogen A alpha and the F8Y mutation. The crystals are in space group P4(3)2(1) 2, with unit-cell dimensions of a = 88.3 Angstrom (1 Angstrom = 0.1 nm ), c = 195.5 Angstrom and two complexes In the asymmetric unit. The fi nal R factor is 0.183 for 2 sigma data from 7.0 to 2.5 Angstrom resolu tion. There is continuous density for the five residues in the P3, P2, P1, P1' and P2' positions of the peptide (Gly-14f to Pro-18f) at the active site of thrombin, and isolated but well-defined density for Tyr -8f at position P9 in the hydrophobic pocket of thrombin. The tyrosine residue is shifted relative to phenylalanine in the native peptide be cause the phenol side chain is larger and makes a novel, intrapeptide hydrogen bond with Gly-14f. Adjacent peptide residues cannot form the hydrogen bonds that stabilize the secondary structure of the native pe ptide. Consequently, the 'reaction' geometry at the scissile bond. eig ht residues from the mutation, is perturbed and the peptide is mostly uncleaved in the crystal structure.