S9, and S10), which had chemically similar amino acids to the ones in M2/M49 or M22/M28 that had been visualized to contact C4BP. structures revealed a uniform and tolerant reading head in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies targeting the M-C4BP interaction, and also inform a path towards vaccine design. Introduction Group A (GAS, a hydrophobic pocket that contains C4BP H67, I78, and L82; a hydrogen bonding group in the form of the main chain nitrogen of C4BP H67; and two positively charged residues, C4BP R64 and R66. Bumetanide The segment that holds this quadrilateral is structurally invariant, being stabilized by a disulfide bond at C65 and limited in conformation by P68 (not depicted). The M proteins supply amino acid side chains that interact with these C4BP residues to form complementary quadrilaterals (Fig. 2b). In all four M-C4BP structures, a hydrophobic M protein residue (usually an aromatic) fits into the hydrophobic pocket, and a polar M protein residue immediately following in sequence hydrogen bonds to the main chain nitrogen of H67. The contacts to C4BP R64 and R66 are predominantly electrostatic (usually salt bridges), but in the case of M49, a polar residue is absent and R64 instead makes hydrophobic contacts, extending its alkyl chains across several M49 residues. These data are compatible with a report that substitution of C4BP residues R64, R66, or H67 with Gln affects binding to M4 and M2222. Decreased affinity results in the case of R64Q and H67Q, but increased affinity occurs for R66Q (likely through a gain-of-function). Open in a separate window Figure 2 C4BP Binding Modea. The C4BP2 quadrilateral (blue dashed lines), with the C4BP2 backbone shown in ribbon representation and key side chains shown as bonds, in which carbons are cyan and nitrogens blue (here and in following panels). The chemical character of M protein residues that interact with the quadrilateral is depicted: , hydrophobic; , negative; H, hydrogen Rabbit Polyclonal to Gastrin bond forming. b. M2, M49, M22, and M28 residues that interact with the C4BP2 quadrilateral and form a complementary quadrilateral (red dashed lines), shown in open-book representation with respect to C4BP2. The M protein backbone is in ribbon representation and key side chains shown as bonds, in which carbons are yellow, oxygens red, and nitrogens blue. The numbering of M proteins is such that the initiator Met is residue 1. c. The C4BP1 Arg39 nook. The depiction and symbols are as for panel a. d. M2, M49, M22, and M28 residues that interact with the C4BP1 Arg39 nook shown in open-book representation. The depiction is as for panel b. Uniform reading head contacts from C4BP1 were far fewer. The key C4BP1 residue was R39, which formed electrostatic contacts through its guanidinium group as well as hydrophobic contacts through its alkyl chain, creating a hydrophobic nook in conjunction with main chain atoms of C4BP1 (Fig 2c). Thus, out of the six C4BP residues that form uniform contacts, three are arginines. This high proportion is likely significant, as the combination of polar and apolar atoms in Arg along with its chain length increase the possibilities for interactions with variable residues. Substitution of C4BP R39 with Gln results in decreased binding to M4 but increased binding to M2222 (again, likely a gain-of-function). All four Bumetanide M proteins have hydrophobic residues that insert into the C4BP1 hydrophobic nook. M2 and M49 also have negatively charged residues that interact with C4BP R39, whereas neither M22 nor M28 do. The importance of C4BP R39 provided an explanation for the aforementioned 180 rotation of C4BP1 (around a hinge at K63, Supplementary Fig. S7). In free C4BP, the C4BP1 R39 nook and the C4BP2 quadrilateral are on opposite sides, and require a 180 reorientation to interact simultaneously with M protein. This 180 rotation was seen in all four structures. However, in one of the two C4BP1-2 molecules bound to M22, the 180 rotation was prevented due to a crystal contact (Supplementary Figs. S7c, d and S8). A similar 180 rotation appears necessary for the interaction of C4BP with C4b, as it has been demonstrated that R39 and the set of residues in C4BP2 that interact with M protein HVRs also interact with C4b22. The purpose of requiring a 180 rotation in C4BP1 to transition Bumetanide between free and bound forms is unclear. Sequence conservation hidden within hypervariability The evidence gathered from these structures proved powerful in bringing to light weak sequence conservation in M protein HVRs. Comparison of the heptad position of M protein residues that interacted with C4BP made it clear that there were two binding.