Hydrophobic residues in the D-domain of a plasminogen-binding M-protein modulate α-helicity, oligomerization, and...

Human plasminogen (hPg) binding M-protein (PAM) is a major virulence determinant of Group A Streptococcus (GAS). PAM contains irregularly spaced heptad repeats, particularly within its C- and D-domains. Our recent cryo-EM structure of PAM from GAS strain AP53 (PAM_AP53) revealed that these repeats do not favor coiled-coil formation, but instead adopt triple-helix bundle and helix-loop-helix motifs in the C- and D-domains, respectively. Interactions between loops connecting helices in these domains maintain the compact conformation of PAM_AP53, correctly orienting human plasminogen (hPg) for activation by the co-expressed streptokinase variant, SK2b. Despite the D-domain being conserved across M-proteins, its specific structural and functional roles remain unknown. To investigate the role of the D-domain in PAM structure-function, we engineered three structure-guided PAM_AP53 variants, replacing nine heptad D-domain residues with glycine (9G), alanine (9A), or leucine (9L). At 25°C, wild-type (WT)-PAM_AP53 exists as a concentration- and temperature-dependent mixture of dimers, tetramers, and higher-order oligomers, which dissociate at physiological temperature. In contrast, PAM_AP53-9A and PAM_AP53-9L form thermostable oligomers while helix-destabilizing PAM_AP53-9G exists as a monomer exhibiting structural loss at all temperatures. All variants retained high hPg-binding affinity. However, unlike WT-PAM_AP53, PAM_AP53-9A, and PAM_AP53-9L, PAM_AP53-9G poorly stimulated hPg activation. Moreover, whereas WT-PAM_AP53, PAM_AP53-9A, and PAM_AP53-9L were covalently attached to the bacterial cell wall and displayed on the cell surface, PAM_AP53-9G, with a significantly reduced helical content, was not exposed on the bacterial surface. Our results reveal the importance of the D-domain to secondary/quaternary structure, attachment of PAM_AP53 to the GAS surface, and SK2b-mediated activation of hPg.