<div><p>(A) Sequence logo of C4BP-binding HVRs. The logo was generated from the seven C4BP-binding HVRs aligned in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B, using WebLogo (<a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a>). Only regions in the HVRs corresponding to the shortest known C4BP-binding region in M22 (residues 1–39) were used to create the logo. These regions are demarcated by the vertical hatched lines in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B. In the logo, each column in the alignment is represented by a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding residue at that position, while the overall height of each stack is proportional to the sequence conservation at that position [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b063" target="_blank">63</a>]. The sequence of the M4.1 HVR was included in the generation of the logo, although it is virtually identical to M4, because the single residue difference between these two HVRs was important for the conclusion that the different HVRs completely lack residue identities (see text). The numbering below the logo refers to residue numbers in the M22 protein and putative coiled-coil heptads (a–g) in M22 are indicated. Asterisks show the position of four M22 residues (L21, E24, L28, and E31) analyzed by site-specific mutagenesis.</p><p>(B) Helical wheel representation of a dimeric coiled-coil [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b042" target="_blank">42</a>]. The sequence of the L21–E31 region of M22 is included, with asterisks above residues L21, E24, L28, and E31, which were analyzed by site-specific mutagenesis and are located within the predicted coiled-coil region. The positions of residues within putative coiled-coil heptads (a–g) are indicated.</p><p>(C–E) The four mutant M22 proteins indicated, constructed by site-specific mutagenesis, and the wild-type (wt) M22 protein, were expressed in <i>S. pyogenes,</i> and the strains were analyzed for surface expression of the proteins and ability to bind C4BP. The genes encoding the proteins were present on plasmid pLZ12Spec, carried by an M-negative strain. This M-negative strain also served as negative control. (C) and (D) show that the different proteins were expressed normally on the bacterial surface (see text). (E) shows that mutants L21A and L28A had completely lost C4BP-binding ability, while mutants E24A and E31A were unaffected. The results shown in (C–E) are based on three separate experiments with duplicate samples and are presented as means ± SD.</p></div
