The plasminogen-binding group A streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine residues

Abstract

The migration of the human pathogen Streptococcus pyogenes (group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the plasminogen-binding group A streptococcal M protein (PAM)-related protein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 microM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (K(d) = 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys(96) and Lys(101) reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg(107) and His(108) to alanine. Furthermore, mutagenesis of Arg(107) and His(108) abolished plasminogen binding by Prp despite the presence of Lys(96) and Lys(101) in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.

FIG. 1.

Phylogeny generated by Bayesian analysis of the amino acid sequences of 13 PAM genes (PAM_NS13, GenBank accession no. AY351851; PAM_NS455, GenBank accession no. AY351857; PAM_NS265, GenBank accession no. AY351855; PAM_NS223, GenBank accession no. AY351854; PAM_NS253, GenBank accession no. AY351853; PAM_NS53, GenBank accession no. AY351852; PAM_NS32, GenBank accession no. AY351850; PAM_NS50.1, GenBank accession no. AY351849; PAM_NS59, GenBank accession no. AY351848; PAM_NS1133, GenBank accession no. AY351847; PAM_NS10, accession no. GenBank AY351846; PAM_NS221, GenBank accession no. DQ136319; NS696 M1 protein, GenBank accession no. AY351858; and Prp, GenBank accession no. AY351856). MLC36 (GenBank accession no. Z32677) and MLC72 (GenBank accession no. Z32678) are plasminogen-binding M protein sequences from group C and group G streptococci, respectively (2). The M1 (11) and NS696 M1 protein sequences were included as outgroups.

FIG. 2.

SDS-PAGE and ligand blot analysis of recombinant M proteins. (A) SDS-12% polyacrylamide gel showing the purified recombinant proteins PAM_NS13 (lane 1), Prp (lane 2), and NS696 M1 (lane 3). Molecular mass markers are given in kilodaltons. (B) Ligand blot analysis employing biotinylated glu-plasminogen of purified recombinant proteins PAM_NS13 (lane 1), Prp (lane 2), and NS696 M1 (lane 3). Molecular mass markers are given in kilodaltons.

FIG. 3.

Saturation binding analysis of biotinylated glu-plasminogen binding to immobilized recombinant proteins. Biotinylated glu-plasminogen binding to immobilized recombinant proteins Prp (A), PAM_NS13 (B), and NS696 M1 protein (C) was measured in the absence (▾, total binding) and presence (⧫, nonspecific binding) of a 50-fold molar excess of unlabeled glu-plasminogen. Specific binding (▪) was determined by subtracting nonspecific binding from total binding at each concentration. A one-site hyperbolic binding function was fitted to the data (P < 0.05), from which the binding dissociation constants were determined. Error bars represent the standard errors of the means.

FIG. 4.

Competition of glu-plasminogen binding to immobilized recombinant Prp with fluid-phase PAM_NS13. Binding of biotinylated glu-plasminogen to immobilized Prp was measured in the presence of various concentrations of unlabeled fluid-phase PAM_NS13. Data points are the mean values of triplicate readings, with error bars indicating standard errors of the means. One-site competition analysis was used to determine the concentration of PAM_NS13 required to inhibit binding of biotinylated glu-plasminogen by 50%.

FIG. 5.

(A) Translated DNA sequences of the plasminogen-binding region (a1/a2 repeats) of the prototype PAM binding site with PAM_NS13 and a putative 21-amino-acid residue Prp plasminogen-binding site. *, residues identical to those of the PAM sequence; -, gaps in the alignment. (B) Alignment of the plasminogen binding domain of wild-type Prp with those of the three site-directed mutants constructed in this study. Mutated residues are indicated in boldface.

FIG. 6.

SDS-PAGE and ligand blot analysis of Prp site-directed mutants. (A) SDS-12% polyacrylamide gel showing the purified recombinant proteins Prp (lane 1), Prp[K⁹⁶K¹⁰¹R¹⁰⁷H¹⁰⁸] (lane 2), Prp[K⁹⁶K¹⁰¹] (lane 3), and Prp[R¹⁰⁷H¹⁰⁸] (lane 4). Molecular mass markers are given in kilodaltons. (B) Ligand blot analysis employing biotinylated glu-plasminogen of purified recombinant proteins Prp (lane 1), Prp[K⁹⁶K¹⁰¹R¹⁰⁷H¹⁰⁸] (lane 2), Prp[K⁹⁶K¹⁰¹] (lane 3), and Prp[R¹⁰⁷H¹⁰⁸] (lane 4). Molecular mass markers are given in kilodaltons.

FIG. 7.

Specific binding of biotinylated glu-plasminogen to immobilized recombinant proteins Prp (▪), Prp[K⁹⁶K¹⁰¹R¹⁰⁷H¹⁰⁸] (*), Prp[K⁹⁶K¹⁰¹] (▾), and Prp[R¹⁰⁷H¹⁰⁸] (•). Specific binding was determined by subtracting nonspecific binding from total binding at each concentration. A one-site hyperbolic binding function was fitted to the data (P < 0.05), from which the binding dissociation constants were determined. Error bars represent the standard errors of the means (n = 3).