Identification of an amino acid signature sequence predictive of protein G-inhibitable IgG3-binding activity in group-A streptococcal IgG-binding proteins

Pathogenic streptococci express surface proteins that bind to host serum proteins. A novel multiple-ligand-binding protein has now been identified in a species belonging to serotype C streptococci. This protein binds to fibrinogen, albumin and IgG and was therefore designated FAI protein. The structure of the fai gene has been determined, and deletion analysis and expression of FAI fusion polypeptides revealed that the binding domain for fibrinogen and IgG is located within the non-repetitive N-terminal half of the protein. A 93-amino acid peptide retained the ability to bind both proteins, whereas a 56-amino acid subpeptide only bound fibrinogen. IgG-binding activity required the complete fibrinogen-binding domain and an additional 37 amino acids C-terminal to it, and albumin-binding activity was only obtained with a polypeptide reflecting the complete surface-exposed region of FAI protein indicating that the binding sites for each ligand were located on overlapping modules. Signal sequence, C repeat region and C-terminus revealed high homology to group A streptococcal M proteins whereas the N-terminal region containing the fibrinogen/IgG-binding domains is completely different and exhibits no similarity to any other previously characterized protein. Thus FAI protein exhibits a framework structure that might have evolved in group C streptococci via fusion of unrelated sequences, thereby generating an albumin-binding domain in the functional context of multiple-ligand-binding activity.

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Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G

Biochemistry ◽

10.1021/bi00147a006 ◽

1992 ◽

Vol 31 (32) ◽

pp. 7243-7248 ◽

Cited By ~ 120

Author(s):

Patrick Alexander ◽

John Orban ◽

Philip Bryan

Keyword(s):

Amino Acid ◽

Kinetic Analysis ◽

Binding Domain ◽

Protein G ◽

Igg Binding ◽

Streptococcal Protein G

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Solubilization of IgG-Binding Proteins from Group A and G Streptococci

Microbiology and Immunology ◽

10.1111/j.1348-0421.1989.tb01504.x ◽

1989 ◽

Vol 33 (2) ◽

pp. 123-127 ◽

Cited By ~ 1

Author(s):

Christopher R. Goward ◽

David A. Barstow

Keyword(s):

Binding Proteins ◽

Igg Binding ◽

Group A

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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

Biochemical Journal ◽

10.1042/bj20030919 ◽

2004 ◽

Vol 381 (3) ◽

pp. 877-885 ◽

Cited By ~ 11

Author(s):

Antonia W. GODEHARDT ◽

Sven HAMMERSCHMIDT ◽

Ronald FRANK ◽

Gursharan S. CHHATWAL

Keyword(s):

Amino Acids ◽

Synthetic Peptides ◽

Binding Protein ◽

Binding Activity ◽

Protein G ◽

Amino Acid Substitutions ◽

Group A Streptococci ◽

Α2 Macroglobulin ◽

Arginine Residues ◽

Group A

GRAB (Protein G-related α2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for α2-macroglobulin (α2M), a broad-range protease inhibitor. It is the sole α2M-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 α2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the α2M-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 α2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the ΔA (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 α2M 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 α2M-binding activity and, thus, their involvement in GRAB-induced virulence.

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Evidence for independent binding domains within a group A streptococcal type IIo IgG-binding protein

Canadian Journal of Microbiology ◽

10.1139/m96-149 ◽

1996 ◽

Vol 42 (11) ◽

pp. 1172-1175 ◽

Cited By ~ 1

Author(s):

Maryanne Tsivitse ◽

Michael D. P. Boyle

Keyword(s):

Amino Acid ◽

Recombinant Protein ◽

Streptococcus Pyogenes ◽

Binding Protein ◽

Mature Protein ◽

Binding Domains ◽

Igg Binding ◽

Group A ◽

Terminal Amino ◽

Internal Fragment

The gene for a type IIo IgG-binding protein has previously been cloned and sequenced. The ~60 000 Mrrecombinant gene product binds all four human IgG subclasses and fibrinogen. Treatment of this recombinant protein with CNBr results in generation of a series of fragments. One fragment, an ~32 000 Mrpolypeptide, binds IgG1, IgG2, and IgG4but neither IgG3nor fibrinogen. N-terminal amino sequencing of this fragment indicated that this was an internal fragment of the protein starting at amino acid 186 of the mature protein. These findings provide evidence for two distinct domains for binding IgG1, IgG2, and IgG4and binding IgG3within a single bacterial IgG-binding protein.Key words: IgG-binding protein, protein H, Streptococcus pyogenes.

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