Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate

Summary. Background: Activated factor XIII (FXIII), a dimer of truncated A-subunits (FXIII-A2*), is a transglutaminase that crosslinks primary amines to peptide-bound glutamine residues. Because in the few natural substrates of FXIII-A2* no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII-A2* is of primary importance. Most of the α2-plasmin inhibitor (α2PI) molecules become truncated by a plasma protease, and the truncated isoform (N1-α2PI) is an important substrate of FXIII-A2*. The crosslinking of N1-α2PI to fibrin plays a major role in protecting fibrin from fibrinolysis. Methods: We studied the interaction of FXIII-A2* with its dodecapeptide glutamine donor substrate, N1-α2PI(1–12), the sequence of which corresponds to the N-terminal sequence of N1-α2PI. Kinetic parameters for N1-α2PI(1–12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1-α2PI(1–12) with FXIII-A2* was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Results and Conclusions: Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII-A2*–N1-α2PI(1–12) complex demonstrated that Asn1 is essential for effective enzyme–substrate interaction. Experiments with C-terminally truncated peptides proved that amino acids 7–12 are essential for the interaction of N1-α2PI(1–12) with the enzyme, and suggested the existence of a secondary binding site on FXIII-A2*. Hydrophobic residues, particularly Leu10 and the C-terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C-terminal residues and FXIII-A2* was demonstrated by STD NMR.