Enhancement through mutagenesis of the binding of the isolated Kringle 2 domain of human plasminogen to omega-amino acid ligands and to an internal sequence of a Streptococcal surface protein

In the background of the recombinant K2 module of human plasminogen (K2(Pg) ), a triple mutant, K2(Pg)[C4G/ E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinit ies of a series of lysine analogs, viz. C-aminobutyric acid, 5-aminopentano ic acid, 4-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyc lohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G ]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximate ly 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence o f up-regulating the lysine binding property of K2(Pg) was explored, as refl ected by its ability to interact with an internal sequence of a plasminogen -binding protein (PAM) on the surface of group A streptococci. A 30-mer pep tide of PAM, containing its K2(Pg)-specific binding region, was synthesized , and its binding to each mutant of K2(Pg) was assessed. Only a slight enha ncement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K-d = 460 nM). A 12-fold decrease in binding affinity was obs erved for K2(Pg)[C4G/L72Y] (K-d = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K-d = 37 nM). Results of these PAM peptide binding studies parallel results of omeg a-amino acid binding to these K2(Pg) mutants, indicating that the high affi nity PAM binding by plasminogen, mediated exclusively through K2(Pg), occur s through its lysine-binding site. This conclusion is supported by the 100- fold decrease in PAM peptide binding to K2(Pg)[C4G/ E56D/L72Y] in the prese nce of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56 ) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupl ed in terms of their contribution to the enhancement of PAM peptide binding .