Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein.
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.
Rios-Steiner
J L
JL
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Schenone
M
M
Mochalkin
I
I
Tulinsky
A
A
Castellino
F J
FJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D006860:Hydrogen Bonding; D018082:Kringles; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D013291:Streptococcus / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D000942:Antigens, Bacterial; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010958:Plasminogen; D008239:Lysine