Structural/Functional Characterization of the α2-Plasmin Inhibitor C-Terminal Peptide
| Autor: | Justin T. Douglas, Michael Locher, Pascal Frank, Miguel Llinás, Johann Schaller |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Plasmin
Stereochemistry Molecular Sequence Data Peptide Serpin Biochemistry Protein Structure Secondary Serine Mice Structure-Activity Relationship Residue (chemistry) Kringles medicine Animals Humans Amino Acid Sequence Nuclear Magnetic Resonance Biomolecular Protein secondary structure Chromatography High Pressure Liquid chemistry.chemical_classification alpha-2-Antiplasmin Circular Dichroism Lysine Plasminogen Ligand (biochemistry) Peptide Fragments Recombinant Proteins Amino acid chemistry Cattle Protein Binding medicine.drug |
| Zdroj: | Biochemistry. 42:1078-1085 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi026917n |
| Popis: | The alpha(2)-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 Asn(398)-Lys(452), 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) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 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(alpha)-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 Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), 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 Lys(452) 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. |
| Databáze: | OpenAIRE |
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