Abstract
The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2hPg) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2hPg/VKK38, with the lysine-binding sites of two K2hPg domains anchored to two regions of monomeric VKK38. The K2hPg/VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2hPg-bound α-helix. The K2hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2hPg Further, the adoption of an α-helix by VKK38 upon binding to K2hPg sterically optimizes the side chains of VKK38 for maximal binding to K2hPg and minimizes steric overlap between the K2hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management.
Highlights
The binding of human plasminogen to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity
The PAM-subtype M protein is present in all pattern D GAS serotypes, and its sequence variability in different GAS isolates provides natural mutations in which to assess the nature of its various functions
One central issue in this study is whether this VHD natural insert would make a significant difference in the hPg-binding pattern and structure of PAM from NS455 (PAM455) compared with PAMs from AP53 (PAM53)
Summary
The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. One subtype of M protein, plasminogen-binding group A streptococcal M-like protein (PAM), has an additional wellknown function of coopting components of the host to evade innate immune responses Important among these properties is the fibrinolytic system, which GAS employs to potently evade innate immunity (e.g. attenuate complement-based opsonization of GAS) [10, 11] and to disseminate into deep tissue. To accomplish this function, PAM first interacts with very high affinity to host human plasma plasminogen (hPg) [12], a step that facilitates its activation to plasmin by GAS-secreted streptokinase [13, 14]. The proteolytic ability of plasmin that is localized on the GAS surface is resistant to natural plasmin inhibitors [13] and is utilized by the bacteria to disrupt barriers to its dissemination (e.g. fibrin that encapsulates GAS) and resonance; ITC, isothermal titration calorimetry; AUC, analytical ultracentrifugation; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane1,3-diol
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