Related Experiment Videos
Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)
Eleni Giannakis1, T Sakari Jokiranta, Rebecca J Ormsby
1Department of Microbiology and Infectious Diseases, Flinders Medical Center, Flinders University, Bedford Park, Adelaide, SA, Australia.
Abstract:
Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.
Insights
Group A Streptococcus (GAS) adheres to skin cells via M protein binding to human CD46 (membrane cofactor protein). This study identifies the M protein binding site on CD46, located in SCRs 3 and 4.
Area of Science:
- Microbiology
- Immunology
- Structural Biology
Background:
- Group A Streptococcus (GAS) adheres to human keratinocytes through an interaction between its M protein and host cell CD46 (membrane cofactor protein).
- CD46, a complement regulator, contains four short consensus repeat (SCR) domains and plays a role in modulating complement activation by C3b/C4b.
- Understanding the specific binding interface is crucial for developing strategies to prevent GAS adherence.
Purpose of the Study:
- To characterize the interaction between streptococcal M protein and the SCR domains of human CD46.
- To identify the precise region of CD46 responsible for M protein binding.
- To determine if the M protein binding site overlaps with the complement regulatory functions of CD46.
Main Methods:
- Confirmation of M6 protein-dependent GAS adherence to keratinocytes.
- Binding assays using soluble recombinant CD46 and CD46 constructs with specific SCR domains.
- ELISA to assess M6 protein binding to CD46 and CD55 chimeras.
- Homology-based molecular modeling of CD46 SCRs 3 and 4.
- Functional assays to evaluate the effect of M6 protein on CD46 cofactor activity and vice versa.
Main Results:
- M6 protein binds to soluble CD46 and a CD46 construct containing SCRs 3 and 4.
- M6 protein binding is abolished when SCRs 3 or 4 are replaced with domains from CD55.
- Molecular modeling suggests a positively charged residue cluster at the interface of SCRs 3 and 4 as the M protein binding site.
- M6 protein does not inhibit CD46 cofactor activity, and C3b does not inhibit M6 protein binding to CD46.
Conclusions:
- GAS adherence to keratinocytes is dependent on the M protein.
- A primary binding site for M protein is located within SCRs 3 and 4 of CD46, likely at their interface.
- This M protein binding site is distinct from the C3b-binding and cofactor site of CD46.