Molecular analysis of Streptococcus pyogenes M1 protein

Streptococcus pyogenes, or group A Streptococcus (GAS), is a common human pathogen that causes a wide spectrum of diseases ranging from mild conditions, such as pharyngitis and impetigo, to life-threatening disease, including streptococcal toxic shock syndrome and necrotizing fasciitis. Antiphagocytic M protein, a cell-wall anchored virulence factor, is expressed by all pathogenic strains of GAS. This molecule is believed to form an alpha-helical coiled coil and demonstrates extensive variability in the surface exposed N-terminus. Binding of host factors such as fibrinogen (Fg), IgG and C4BP by M protein confers antiphagocytic properties to Gas. The X-ray crystallographic structure of an N-terminal fragment of M1 protein is presented here, revealing a disordered N- terminus followed by ordering of the A-region into a dimeric alpha-helical coiled coil. The Fg-binding B- repeats have disrupted coiled-coil packing, causing the alpha-helices in this region to splay apart from one another. The disrupted coiled-coil packing leads to instability of the M1 oligomeric state. Static light scattering and circular dichroism show that the M1 fragment exists in a dynamic equilibrium between monomers and dimers. The oligomeric state is especially sensitive to temperature; however, binding assays demonstrate that fibrinogen binding is independent of temperature. Structural analysis of the B-repeats showed that it contains many unfavorable residues in its a and d positions. These residues were mutated to optimal coiled- coil packing residues to determine the significance of M1 protein conserving non-canonical residues in these critical positions. Surprisingly, the optimized construct formed trimeric species and abrogated Fg-binding. The unexpected oligomeric state in the mutant constructs could be partially converted back to the monomer/dimer population observed in the native sequence by deleting a single amino acid just prior to the B-repeats. These species also did not bind Fg and indicated that the residues presiding in the core packing positions in the B- repeats may be critical in contacting Fg. We propose the B -repeats maintain a splayed state in the mature protein presented on the GAS surface and that this conformation is required to expose the residues that would otherwise be involved in coiled-coil packing. Instead these residues form the Fg-binding site in M1 protei