title: 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: A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.
authors: From the W. M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and.
authors: Sarbani
authors: S
authors: Bhattacharya
authors: From the W. M. Keck Center for Transgene Research and.
authors: Zhong
authors: Z
authors: Liang
authors: the Department of Biochemistry and Molecular Biology, Monash University, 3800, Victoria, Australia.
authors: Adam J
authors: AJ
authors: Quek
authors: From the W. M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and.
authors: Victoria A
authors: VA
authors: Ploplis
authors: the Department of Biochemistry and Molecular Biology, Monash University, 3800, Victoria, Australia.
authors: Ruby
authors: R
authors: Law
authors: From the W. M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and fcastell@nd.edu.
authors: Francis J
authors: FJ
authors: Castellino
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation; D013297:Streptococcus pyogenes / Q000378:metabolism / Q000472:pathogenicity*; D014461:Ultracentrifugation; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D010958:Plasminogen
keywords: Bacteria; Bacterial Pathogenesis; Protein Domain; Protein Engineering; Protein Folding