Binding of the kringle‐2 domain of human plasminogen to streptococcal PAM‐type M‐protein causes dissociation of PAM dimers

Abstract

The direct binding of human plasminogen (hPg), via its kringle‐2 domain (K2 hPg ), to streptococcal M‐protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAM NS88.2 ), with one K2 hPg binding a‐repeat in its A‐domain, we employed biophysical techniques to analyze the mechanism of the K2 hPg /PAM NS88.2 interaction. We show that apo‐PAM NS88.2 is a coiled‐coil homodimer (M.Wt. ~80 kDa) at 4°C–25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature‐dependent dissociation of PAM NS88.2 over a narrow temperature range. PAM NS88.2 displayed a single tight binding site for K2 hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2 hPg /PAM NS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2 hPg /PAM NS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAM NS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2 hPg ) showing that PAM is a functional monomer at 37°C.

A high-resolution cryo-EM structure of a bacterial M-protein reveals a compact structure that diverges from related M-proteins

“…While we could not resolve hPg to the same high degree as that of PAM AP53 ( Supplementary Fig. S3 ), we nonetheless were able to map the known region of hPg (8, 11, 13, 33–35) that was bound to PAM by placing the 3D structure of the hPg-K2 domain within the confines of the experimentally-determined map coordinates.…”

“…Other subgroups of M-Prts have been shown to bind to fibrinogen directly (7) which then recruits hPg to the surface where it is activated to hPm. PAM-type M-Prts have been shown to have the highest binding affinity for hPg (K d ∼1 nm) (8) when compared to other putative receptors within GAS cells, viz., α-enolase (SEN) or GAPDH (9, 10). This high binding affinity, natural protection, and high surface abundance makes PAM-type M-Prts an important target for research in understanding the interactions between hPg and GAS cells that result in its pathogenicity.…”

Plasminogen missense variants and their involvement in cardiovascular and inflammatory disease

“…This predicted pathogenic variants would affect the stability of the PLG three-dimensional structure. We found that this variant was easier to activate, most likely due to a more relaxed PLG conformation with increased exposure of the Arg 561 -Val 562 activation cleavage site ( 118 ). This variant should also facilitate proteolysis and reduce the half-life of PLG in vivo, which is consistent with PDI.…”

“…In a study of the interaction of PLG with Group A Streptococcus pyogenes (GAS) surface proteins in vitro ( 32 , 118 ), our group has expressed and characterized the PDI-associated missense variant PLG/D 219 N that affects the K2-LBS anionic ligand binding region. Unlike WT-PLG, PLG/D 219 N precludes GAS from effective invasion by defective interaction with the cell surface PLG binding protein, plasminogen-binding group A streptococcal M-protein (PAM).…”

“…Carriers of this PDI variants may have a competitive advantage against S. pyogenes infection . In PLG/D 219 N, the intramolecular salt bridge between K2-PLG and Lys 708 of the SP residue is disrupted ( 32 , 118 ). This predicted pathogenic variants would affect the stability of the PLG three-dimensional structure.…”