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 (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha 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 (kcat/Km) toward hydrolysis of fibrinogen Aalpha(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.
Maurer
M C
MC
Baker Laboratory of Chemistry, Cornell University Biotechnology Resource Center, Ithaca, New York 14853-1301, USA.
Peng
J L
JL
An
S S
SS
Trosset
J Y
JY
Henschen-Edman
A
A
Scheraga
H A
HA
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D005344:Fibrinopeptide A / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011522:Protons; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
D000577:Amides; D011522:Protons; D005344:Fibrinopeptide A; D013917:Thrombin