Identification of the Streptococcal M Protein Binding Site on Membrane Cofactor Protein (CD46)

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.

Structure of the Extracellular Portion of CD46 Provides Insights into Its Interactions with Complement Proteins and Pathogens

“…Regions that interact with both viruses are highlighted in black. Residues predicted to contact the Streptococcus M protein (M-prot) [38] are shown in purple.…”

“…Notably, the regions of CD46 that are thought to interact with C3b and C4b overlap but are not identical [37]. As the cellular C3b and C4b proteins as well as HHV6 engage regions that include the SCR3 and/or SCR4 domains, modeling studies have aimed to predict the structure of unknown portions of CD46 in order to provide a basis for the mapping of binding epitopes [37], [38]. Although some features of the SCR domains are conserved and can be predicted with reasonable accuracy, loop regions and interdomain orientations are notoriously difficult to model.…”

CD46: expanding beyond complement regulation

“…Although fine mapping of these sites has not been performed, domains necessary to these interactions have been identified. Pilus-mediated adhesion of Neisseria gonorrhoeae and interactions with the streptococcal M protein both require regions in CCPs 3 and 4, whereas human herpesvirus 6 requires CCPs 2 and 3 [41][42][43][44][45]. Engagement of CD46 by pathogens has implications beyond cellular adhesion.…”

Gram—Positive Adhesins

“…The carboxy-terminal region of M6 protein as well as the short consensus domains 3 and 4 of CD46 were shown to be crucial for M6/CD46-mediated keratinocyte attachment [40]. Although structurally closely related, M proteins represent a heterogeneous group of adhesins with respect to their ligands or target cells [41].…”