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Identification of a plasminogen-binding motif in PAM, a bacterial surface protein
A C Wistedt1, U Ringdahl, W Müller-Esterl
1Department of Medical Microbiology, University of Lund, Sweden.
Abstract:
Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.
Insights
Group A Streptococcus M-like proteins bind plasminogen via lysine-rich repeats. These repeats are crucial for plasminogen interaction, offering insights into bacterial invasion mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Surface-associated plasminogen contributes to cellular invasion.
- Identifying plasminogen-binding surface proteins is key to understanding this process.
Purpose of the Study:
- To clone and express N-terminal variable regions of M-like (ML) proteins from Group A Streptococcus.
- To analyze the plasminogen-binding properties of these recombinant ML proteins.
Main Methods:
- Cloning and expression of ML protein N-terminal regions in E. coli.
- Plasminogen binding assays, including inhibition studies with 6-aminohexanoic acid.
- Sequence analysis and experiments with fusion proteins and synthetic peptides to map binding sites.
Main Results:
- Recombinant ML proteins from five serotypes bound plasminogen with high affinity.
- Binding involved plasminogen kringle domains and was mediated by lysine residues in ML proteins.
- Tandem repeats with central lysine residues were identified as major plasminogen and tissue-type plasminogen activator binding sites.
- Lysine to alanine substitution in ML53 protein reduced plasminogen binding by 80%.
Conclusions:
- Lysine-rich tandem repeats in M-like proteins are critical for plasminogen binding.
- These findings precisely localize the binding domain, providing a tool for studying kringle-protein interactions.