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 affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-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 approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected 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 peptide 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 enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed 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 omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs 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 presence 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 coupled in terms of their contribution to the enhancement of PAM peptide binding.
Nilsen
S L
SL
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Prorok
M
M
Castellino
F J
FJ
D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism*; D006801:Humans; D018082:Kringles / Q000235:genetics*; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives*; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013291:Streptococcus
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; C084529:plasminogen-binding protein, bacteria; D010958:Plasminogen; D008239:Lysine