A study of structural motifs of plasminogen and the interaction of its second kringle domain with a surface -exposed streptococcal protein, PAM

The cleavage site loop of plasminogen has been investigated and modulation of its susceptibility to different plasminogen activators studied using mutated proteins and the corresponding sequences in isolated synthetic peptides. Site directed mutagenesis and basic kinetic techniques demonstrate that the properties of isolated peptides do not always correspond to the same behaviour in full-length proteins. The specificity of plasminogen activators for the cleavage site loop is dependent not only upon the primary sequence but elements present in the full-length protein as well. In this study it was shown that isolated peptides related to the plasminogen cleavage site loop may be more susceptible to cleavage by plasminogen activators than peptides with the primary sequence of the cleavage site loop, but in the full-length protein, those activators do not cleave these sequences. In a second study the determinants of binding of plasminogen kringle 2 and P&barbelow;lasminogen binding group A&barbelow; streptococcal M&barbelow;-like protein (PAM) were investigated using an isolated kringle 2 molecule designed to have an upregulated lysine binding site: K2 pg[C4G/E56D/L72Y] and a synthetic 30 residue peptide (VEK 30) corresponding to the primary sequence within PAM protein shown to be important for binding, as well as truncation peptides derived from VEK 30. The importance of this interaction resides in its involvement in contributing to bacterial dissemination by virtue of the ability of PAM to bind to HPg, and thus form surface-bound plasmin (Pm) with the aid of the endogenous bacterial Pg activator, streptokinase (SK). Complementary approaches were undertaken to identify residues important for binding: NMR chemical shift analysis, isothermal titration calorimetry (ITC) for evaluation of binding constants of ligands, and a surface-enhanced-laser-desorption-ionization time-of-flight mass spectrometry (SELDI)-based screening assay for definition of variant peptides that interacted with K2pg[C4G/E 56D/L72Y]. Predictions made by interpretation of the biochemical and biophysical data was corroborated by the crystal structure of the K2pg[C4G/E56D/L72Y]/VEK 30 complex. Arg17, His18 and Glu20 residues from PAM are found in the LBS of K2pg[C4G/E 56D/L72Y].