Structural/Functional Characterization of the α2-Plasmin Inhibitor C-Terminal Peptide

Abstract

The α₂-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn³⁹⁸−Lys⁴⁵², and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K_a ∼ 69.9 mM^-1), K4 (K_a ∼ 45.7 mM^-1), K5 (K_a ∼ 4.3 mM^-1), and r-K2 (K_a ∼ 3.2 mM^-1), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N^α-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys⁴⁵² plays a major role in the binding, internal residues in r-A2PIC tether the kringles. ¹H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp²⁵, Trp⁶², Phe⁶⁴, Trp⁷², and Tyr⁷⁴, selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys⁴⁵² most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.