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, K2(Pg)[C(4)G/(ED)-D-56/L(72)y] (MK2(Pg)), co ntaining an upregulated lysine-binding site, bound to a functional 30 resid ue internal peptide (VEK-30) from an M-type protein of a group A Streptococ cus surface protein, has been determined by molecular replacement methods u sing K4(Pg) as a model, and refined at 2.7 Angstrom resolution to a X-facto r of 19.5 %. The X-ray crystal structure shows that VEK-30 exists as a near ly end-to-end alpha -helix in the complex with mK2(Pg). The final structure also revealed that Arg17 and His18 of VEK-30 served as cationic loci for A sp54 and Asp56 of the consensus lysine-binding site of mK2(Pg), while Glu20 of VEK-30 coordinates with Arg69 of the cationic binding site of mK2(Pg). The hydrophobic ligand-binding pocket in mK2(Pg), consisting primarily of T rp60 and Trp70, situated between the positive and negative centers of the l ysine-binding site, is utilized in a novel manner in stabilizing the intera ction with VEK-30 by forming a cation-pi -electron-mediated association wit h the positive side-chain of Arg17 of this peptide. Additional lysine-bindi ng sites, as well as exosite electrostatic and hydrogen bonding interaction s involving Glu9 and Lys14 of VEK-30, were observed in the structural model . The importance of these interactions were tested in solution by investiga ting the-binding constants of synthetic variants of VEK-30 to mK2(Pg), and it was found that, Lys14, Arg17, His18, and Glu20 of VEK-30 were the most c ritical amino acid binding determinants. With regard to the solution studie s, circular dichroism analysis of the titration of VEK-30 with mK2(Pg) demo nstrated that the peptidic a-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 inter action of the lysine-binding site of a kringle with an internal pseudo-lysi ne residue of a peptide or protein that functionally interacts with a kring le module, and serves as a paradigm for this important class of interaction s. (C) 2001 Academic Press.