An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2(pg)[C(4)G/(ED)-D-56/(KY)-Y-72]) Of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of al-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His(12) in the peptide/protein complex. In an effort to screen al-PAM-derived truncation peptides, a combinatorial mixture, a1 Delta a2-PAM[(HX)-X-12] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2(pg)[C(4)G/(ED)-D-56/(KY)-Y-72]-containing chip was that containing His(12) corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys(12)-, Arg(12)- and Trp(12)- containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.