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(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)/PAK(NS88.2) interaction. We show that apo-PAM(NS8)(8.)(2) is a coiled-coil homodimer (M.Wt. similar to 80 kDa) at 4 degrees C-25 degrees C, and is monomeric (M.Wt. similar to 40 kDa) at 37 degrees 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 degrees C, which progressively increased at 25 degrees C through 37 degrees C. We isolated the K2(hPg)/PAM(NS88)(.2) complexes at 4 degrees C, 25 degrees C, and 37 degrees C and found molecular weights of similar to 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 degrees C.