Conformationally organized lysine isosteres in Streptococcus pyogenes M protein mediate direct high-affinity binding to human plasminogen

Abstract

The binding of human plasminogen (hPg) to the surface of the human pathogen group A (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 (K2) 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 K2 with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2/VKK38, with the lysine-binding sites of two K2 domains anchored to two regions of monomeric VKK38. The K2/VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2-bound α-helix. The K2 residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2 We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2 Further, the adoption of an α-helix by VKK38 upon binding to K2 sterically optimizes the side chains of VKK38 for maximal binding to K2 and minimizes steric overlap between the K2 domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management.

Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

“…Escherichia coli BL21/DE3 cells (New England BioLabs) were transformed with different recombinant expression plasmids (pET-28a/pET-15b). The procedure for protein expression has been previously described (Yuan et al, 2017). In order to increase the yields, terrific broth medium (TB; 12 g tryptone/24 g yeast extract/4 mL glycerol/2.3 g KH 2 PO 4 /12.5 g K 2 HPO 4 per liter) was applied instead of conventional Luria-Bertani (LB) medium.…”

“…Accordingly, the flow-through fraction contained those short PAM-derivative peptides. The protease inhibitor, p-aminobenzamidine, immobilized on agarose (Sigma), was added to the aforementioned fraction to remove thrombin (Yuan et al, 2017) and the resin subsequently removed by centrifugation.…”

“…Different concentrations of analytes in HBS-EP buffer were injected over the hPg-bound CM5 chip surface for an association time of 120 sec and a dissociation time of 360 sec (Qiu et al, 2018;Yuan et al, 2017). The gold surface was regenerated using 10 mM glycine, pH 1.5.…”

“…Due to the lack of the RH2-motif in the VEK30 AP53 , interactions between the a2-repeatcontaining region and K2 hPg are absent (Wang et al, 2010a;Wang et al, 2010b). However, considering the high homology between a1and a2-repeats in PAM AP53 , the residues in each tandem repeat involved in its binding to K2 hPg are likely similar (Yuan et al, 2017).…”

Toward Better Understanding on How Group AStreptococcusManipulates Human Fibrinolytic System

“…In bacterial strains such as PAM NS265 and PAM NS32 , the second RH motif is mutated to Arg-Tyr and Gly-His, respectively (Figure 5a). Despite these variations, all PAM bind to human Plg with high affinities [71,73,74].…”