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 (PAMNS88.2), with one K2hPg binding
a-repeat in its A-domain, we employed biophysical techniques to analyze
the mechanism of the K2hPg/PAMNS88.2 interaction. We show that
apo-PAMNS88.2 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 PAMNS88.2 over a
narrow temperature range. PAMNS88.2 displayed a single tight binding
site for K2hPg at 4°C, which progressively increased at 25°C through
37°C. We isolated the K2hPg/PAMNS88.2 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) K2hPg/PAMNS88.2 monomer
complex. hPg activation experiments by streptokinase demonstrated that
the hPg/PAMNS88.2 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 (K2hPg)
showing that PAM is a functional monomer at 37°C.
NMR Backbone Dynamics of VEK-30 Bound to the Human Plasminogen Kringle 2 Domain
