Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein
| Autor: | J L, Rios-Steiner, M, Schenone, I, Mochalkin, A, Tulinsky, F J, Castellino |
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| Rok vydání: | 2001 |
| Předmět: |
Models
Molecular Antigens Bacterial Binding Sites Circular Dichroism Lysine Molecular Sequence Data Static Electricity Streptococcus Hydrogen Bonding Plasminogen Calorimetry Crystallography X-Ray Ligands Peptide Fragments Protein Structure Secondary Recombinant Proteins Amino Acid Substitution Kringles Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Humans Thermodynamics Amino Acid Sequence Protein Binding |
| Zdroj: | Journal of molecular biology. 308(4) |
| ISSN: | 0022-2836 |
| Popis: | The X-ray crystal structure of a complex of a modified recombinant kringle-2 domain of human plasminogen, K2Pg[C4G/E56D/L72Y] (mK2Pg), containing an upregulated lysine-binding site, bound to a functional 30 residue internal peptide (VEK-30) from an M-type protein of a group A Streptococcus surface protein, has been determined by molecular replacement methods using K4Pg as a model, and refined at 2.7 A resolution to a R-factor of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a nearly end-to-end alpha-helix in the complex with mK2Pg. The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for Asp54 and Asp56 of the consensus lysine-binding site of mK2Pg, while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2Pg. The hydrophobic ligand-binding pocket in mK2Pg, consisting primarily of Trp60 and Trp70, situated between the positive and negative centers of the lysine-binding site, is utilized in a novel manner in stabilizing the interaction with VEK-30 by forming a cation-pi-electron-mediated association with the positive side-chain of Arg17 of this peptide. Additional lysine-binding sites, as well as exosite electrostatic and hydrogen bonding interactions involving Glu9 and Lys14 of VEK-30, were observed in the structural model. The importance of these interactions were tested in solution by investigating the binding constants of synthetic variants of VEK-30 to mK2Pg, and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most critical amino acid binding determinants. With regard to the solution studies, circular dichroism analysis of the titration of VEK-30 with mK2Pg demonstrated that the peptidic alpha-helical structure increased substantially when bound to the kringle module, in agreement with the X-ray results. This investigation is the first to delineate structurally the mode of interaction of the lysine-binding site of a kringle with an internal pseudo-lysine residue of a peptide or protein that functionally interacts with a kringle module, and serves as a paradigm for this important class of interactions. |
| Databáze: | OpenAIRE |
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