Structural Examination of the Influence of Phosphorylation on the Binding of Fibrinopeptide A to Bovine Thrombin

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (AαBβγ)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aα chain, cleavage occurs most prevalently at the Arg16−Gly17 peptide bond. About 25−30% of the human fibrinogen Aα chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme−substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (k cat/K m) toward hydrolysis of fibrinogen Aα(1−20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1−5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1−16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.