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

The direct binding of human plasminogen (hPg), via its kringle-2 domain (K2 ), 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 ), with one K2 binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2 /PAM interaction. We show that apo-PAM 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 over a narrow temperature range. PAM displayed a single tight binding site for K2 at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2 /PAM 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 /PAM  monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAM  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 ) showing that PAM is a functional monomer at 37°C.