The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen- binding group A streptococcal M- like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site- directed mutagenesis of full- length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys(98) and Lys(111)) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg(101) and His(102) and Glu(104), which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg(114) and His(115) to alanine. Furthermore, mutagenesis of both arginine (Arg(101) and Arg(114)) and histidine (His(102) and His(115)) residues abolished interaction with plasminogen despite the presence of Lys(98) and Lys(111) in the binding repeats. This study shows for the first time that residues Arg(101), Arg(114), His(102), and His(115) in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.