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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
- Source :
- The Biochemical journal. 381(Pt 3)
- Publication Year :
- 2004
-
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.
- Subjects :
- DNA, Bacterial
Arginine
Streptococcus pyogenes
Virulence Factors
Amino Acid Motifs
Molecular Sequence Data
Peptide
Biochemistry
Binding, Competitive
Residue (chemistry)
Bacterial Proteins
Protein Interaction Mapping
Humans
alpha-Macroglobulins
Amino Acid Sequence
Binding site
Cloning, Molecular
Molecular Biology
chemistry.chemical_classification
Binding Sites
biology
Binding protein
Cell Biology
Peptide Fragments
Amino acid
Protein Structure, Tertiary
chemistry
biology.protein
Mutagenesis, Site-Directed
Protein G
Carrier Proteins
Peptides
Binding domain
Protein Binding
Research Article
Subjects
Details
- ISSN :
- 14708728
- Volume :
- 381
- Issue :
- Pt 3
- Database :
- OpenAIRE
- Journal :
- The Biochemical journal
- Accession number :
- edsair.doi.dedup.....b39df6376b30b43a6cbfacc4becce6b7