Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

Abstract

Background: High affinity human plasminogen (hPg) binding to Group A Streptococcus (GAS) is mediated by the coiledcoil M-like protein, PAM. Results: Amino acid residues in the a1a2 domain of PAM direct high affinity PAM/hPg binding regardless of oligomerization status of PAM. Conclusion: The a1a2 domain defines its hPg binding site, regardless of PAM dimerization. Significance: The virulence of GAS is altered by mutations in PAM(a1a2).

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

“…The binding affinities of full-length PAMs to hPg or of VEK/ VKK peptides (listed in Table 1) to K2 hPg were assessed by surface plasmon resonance (SPR). The prototype PAM53 has strong binding ability to hPg, with a K D of ϳ1 nM, consistent with previous results (12). Despite PAM455 containing a naturally occurring 121 VHD 123 motif immediately following the RH dipeptide in the a1 repeat (RH1), no effects on the binding affinity of PAM455 to hPg were found ( Table 2).…”

“…b The residues of the binding sites are italicized, and the VHD insertions are shown in bold. c Truncated peptides published previously (12) (Table 3). VEK30/ 19 VHD 21 exhibits a high dissociation rate; thus, we confirmed its K D to K2 hPg using the SPR affinity method.…”

“…However, no structure for this protein is available despite its pathological significance. PAM was previously shown to be a dimer (12,32). Two RH motifs, located in flexible region of the a1a2 domain of PAM, play important roles in its binding to host hPg.…”

“…However, the K D of this interaction was ϳ30 -50-fold weaker than that of the PAM53-K2 hPg complex. A redesign of VEK30 resulting from an addition of two C-terminal amino acids ( 30 RH 31 ) from PAM53 or five C-terminal amino acids ( 30 RHDHD 34 ) from PAM53 (Table 1) strengthened the binding affinity of the resulting peptides (VEK32 and VEK35, respectively) to that of full-length PAM53 (12). We then decided to assess the role of these additional amino acids in a1a2 binding to K2 hPg via functional and highresolution structural analyses.…”

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein

“…4B). On the other hand hPg binds to rPAM with a K d of ϳ1 nM (43), and hFg interacts with rM1 with a K d of ϳ4 nM (38). Considering the mouse resident proteins, the binding of mFg to rM1 was also measured.…”