The Lack of Binding of VEK-30, an Internal Peptide from the Group A Streptococcal M-like Protein, PAM, to Murine Plasminogen Is due to Two Amino Acid Replacements in the Plasminogen Kringle-2 Domain

Abstract

VEK-30, a 30-amino acid internal peptide present within a streptococcal M-like plasminogen (Pg)-binding protein (PAM)from Gram-positive group-A streptococci (GAS), represents an epitope within PAM that shows high affinity for the lysine binding site (LBS) of the kringle-2 (K2) domain of human (h)Pg. VEK-30 does not interact with this same region of mouse (m)Pg, despite the high conservation of the mK2-and hK2-LBS. To identify the molecular basis for the species specificity of this interaction, hPg and mPg variants were generated, including an hPg chimera with the mK2 sequence and an mPg chimera containing the hK2 sequence. The binding of synthetic VEK-30 to these variants was studied by surface plasmon resonance. The data revealed that, in otherwise intact Pg, the species specificity of VEK-30 binding in these two cases is entirely dictated by two K2 residues that are different between hPg and mPg, namely, Arg-220 of hPg, which is a Gly in mPg, and Leu-222 of hPg, which is a Pro in mPg, neither of which are members of the canonical K2-LBS. Neither the activation of hPg, nor the enzymatic activity of its activated product, plasmin (hPm), are compromised by replacing these two amino acids by their murine counterparts. It is also demonstrated that hPg is more susceptible to activation to hPm after complexation with VEK-30 and that this property is greatly reduced as a result of the R220G and L222P replacements in hPg. These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM ؉

Novel Conserved Group A Streptococcal Proteins Identified by the Antigenome Technology as Vaccine Candidates for a Non-M Protein-Based Vaccine

“…Many different surface molecules mediate the attachment of GAS to human extracellular matrix components, including fibronectin, fibrinogen, laminin, and collagen, and the interaction with human plasma proteins (33,62). Among these streptococcal surface proteins, M protein binds to fibrinogen and plasminogen (50,54), the adhesin protein F binds to fibronectin (26,46), Lbp has been described to bind laminin (60), PAM binds to plasminogen (3,22), and Cpa binds to type I collagen (34,64). Based on in vitro binding assays, we observed that deletion of the spy1536 gene resulted in a drastically reduced interaction of S. pyogenes with all human extracellular matrix proteins tested.…”

Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)

“…Additionally, the finding that the binding of VEK-30 to K1-K3 results in the disappearance of K3 electron density [13] suggests that a large conformational change may be operative in Pg-PAM functionality, akin to that observed for Pg in the presence of small-molecule activators such as 6-aminohexanoic acid (6-AHA) [14, 15]. Preliminary data from a recent study, in which VEK-30 was found to augment SK-mediated activation of Pg, is likewise supportive of this premise [16]. The present study was thus undertaken to determine if structural alterations attend the binding of VEK-30 to Pg, as evaluated by the effects of the bacterial peptide on the hydrodynamic volume and activation rate of Pg.…”

A Natural Inactivating Mutation in the CovS Component of the CovRS Regulatory Operon in a Pattern D Streptococcal pyogenes Strain Influences Virulence-associated Genes*

“…The a1(italics)/a2(boldface) locus, which contains the hPgbinding site in PAM AP53 (amino acids sufficient and critical for hPg binding are underlined) (19,(37)(38)(39), is not present in EMM NS931 , thus rendering this M-protein incapable of directly binding to hPg.…”