Binding of α2-macroglobulin to GRAB (protein G-related α2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the ΔA region

GRAB (Protein G-related a,M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for a, macroglobulin (a,M), a broad-range protease inhibitor. It is the sole alpha(2)M-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 alpha(2)M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha(2)M-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 a(2)M-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 PRIIPN-GGTL (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 alpha(2)M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg(42) --> Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha(2)M-binding activity and, thus, their involvement in GRAB-induced virulence.