The Gram-positive bacterium Streptococcus pyogenes, known as group A Streptococcus (GAS), is a widespread human pathogen responsible for numerous disease manifestations, ranging from mild infections to severe invasive diseases and major autoimmune sequelae. At present there is no vaccine against GAS, with one of the major impediments being the antigenic variability of the M protein, one of the primary surface-associated virulence factors of GAS. The M protein elicits protective opsonizing antibodies, but these antibodies generally target the M protein hypervariable region (HVR) and accordingly the resulting immunity is specific to a particular M protein antigenic variant and does not extend to other M protein antigenic variants. With more than 200 distinct HVRs having been identified, the difficulty in developing a vaccine that offers universal coverage is apparent.

In addition to providing antigenic variation, the M protein inhibits phagocytic uptake and clearance of GAS by neutrophils and macrophages through the recruitment of human C4b-binding protein (C4BP), a negative regulator of the complement system. C4BP is bound by the M protein HVR. In stark contrast to the strict specificity of antibodies, C4BP recognizes a remarkably broad range of M protein HVRs (~88% in one study). This suggests that a structural understanding the C4BP-HVR interaction may be applicable to the design of broadly neutralizing antibodies.

To achieve the structural understanding necessary to address the obstacle of antigenic variability in GAS vaccine design, the co-crystal structures of multiple M protein HVRs (M2HVR, M22HVR, M49HVR, and M28HVR) in complex with the (C4BP) domains responsible for binding (C4BPα1-2) were determined. A comparative analysis of these co-crystal structures suggests a conserved C4BP binding mode. Based on the heptad position of interacting M protein residues, two distinct conserved M protein binding motifs were also identified. Structural observations were tested through a series of binding studies of M protein substitution mutants. These results identified M protein HVR residues necessary for C4BP binding as well as give evidence of a remarkable amount of tolerance at the HVR ‘reading head’. Overall, this analysis offers extensive structural insight into how C4BP recognizes a broad range of M protein HVRs, which potentially has direct implications in GAS vaccine design.