Streptococcal M protein and human C4BP

Sammanfattning: Antigenic variation of surface proteins allows microorganisms to evade the immune system of the infected host. This phenomenon represents an apparent paradox, because the variable protein must retain an important function, while its antigenic properties vary extensively. The surface associated M protein of Streptococcus pyogenes, a common human pathogen, exhibits antigenic variation due to an N-terminal hypervariable region (HVR). The HVRs of many M proteins bind the human complement regulator C4b-binding protein (C4BP), which down-regulates deposition of complement on the bacterial surface and thereby protects the bacteria against complement-mediated phagocytosis. Different immunological, biochemical and structural aspects of this biologically important interaction is the focus of the four papers included in this thesis. C4BP-binding HVRs exhibit remarkable sequence divergence, yet bind the same ligand. In the first study, we found that such HVRs can be studied in isolated form, as synthetic peptides, thus allowing us to directly characterize the HVRs and their interaction with C4BP. Our data indicate that the peptides bind to the same region in C4BP and assume similar folds, although they are antigenically unrelated. In the second study, we show that such a synthetic peptide can be used to purify human C4BP by a simple one-step affinity-chromatography method. The third study was focused on the sequence divergence among C4BP-binding HVRs. Remarkably, analysis of seven HVRs demonstrated that they completely lack residue identities. However, use of site-specific mutagenesis to substitute relatively conserved residues in the M22 protein indicated that the predicted coiled-coil structure of the HVR is crucial for ability to bind C4BP. Interestingly, change of single residues that do not affect C4BP-binding induced major immunological changes. Together, the data in paper III indicate that HVRs of C4BP-binding M proteins have an extraordinary capacity for sequence change and antigenic variability, while retaining a specific ligand-binding function. In paper IV, we studied the three-dimensional structure of C4BP-binding HVRs, using peptides derived from the M4 and M22 proteins. No structure could be obtained, but the data clearly indicate that the central parts of the HVRs are folded as coiled-coils, both in solution and in complex with C4BP, while the termini are flexible. Remarkably, the peptides derived from M4 and M22 appear to adopt similar structures, in spite of a limited number of residue identities.