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

• Published in: Journal of thrombosis and haemostasis 2009-04, Vol.7 (4), p.627-633
• Main Authors: PÉNZES, K., KÖVÉR, K. E., FAZAKAS, F., HARAMURA, G., MUSZBEK, L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Asparagine; Binding Sites; Cattle; Factor XIIIa - metabolism; factor XIII; factor XIII substrate peptides; Glutamine - metabolism; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Magnetic Resonance Spectroscopy; Mutation; Peptides - metabolism; Protein Binding; STD NMR; Substrate Specificity; transglutaminase; α2‐plasmin inhibitor
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2009.03291.x

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.