title: Binding of alpha2-macroglobulin to GRAB (Protein G-related alpha2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the DeltaA region.
abstract: GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42-->Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.
authors.affiliation: Department of Microbial Pathogenesis and Vaccine Research, GBF-German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
authors.forename: Antonia W
authors.initials: AW
authors.lastname: Godehardt
authors.forename: Sven
authors.initials: S
authors.lastname: Hammerschmidt
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.forename: Gursharan S
authors.initials: GS
authors.lastname: Chhatwal
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D013297:Streptococcus pyogenes / Q000235:genetics / Q000472:pathogenicity*; D037521:Virulence Factors / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; C119608:GRAB protein, Streptococcus pyogenes; D010446:Peptide Fragments; D010455:Peptides; D037521:Virulence Factors; D000511:alpha-Macroglobulins; D001120:Arginine