C Repeats of the Streptococcal M1 Protein Achieve the Human Serum Albumin Binding Ability by Flanking Regions Which Stabilize the Coiled-Coil Conformation

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Abstract
The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the α-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an α-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 °C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of ∼31 and ∼65 °C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower α-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of α-helical single strands only below 20 °C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

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