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.