The Binding Site for Human C4BP in the Hypervariable Region (HVR) of M Protein

Abstract

<div><p>(A) Schematic representation of C4BP bound to the HVR of an M protein, a dimeric coiled-coil. The most common form of C4BP has seven identical α-chains and one short β-chain. Both chains are composed of CCP modules, as indicated. The binding site for M protein in C4BP is located in the CCP1–2 region of the α-chain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b017" target="_blank">17</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b047" target="_blank">47</a>].</p><p>(B) Multiple sequence alignment of HVRs that bind C4BP. The five upper sequences are from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b025" target="_blank">25</a>]. Three residues that are identical in these five sequences are boxed. PrtH is a second M protein expressed by certain M1 strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b035" target="_blank">35</a>]. The lower part of the alignment shows the HVRs of M4.1 and M114, characterized in this paper. The vertical hatched lines, corresponding to residues 1–39 in M22, indicate the region used to generate the logo in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g005" target="_blank">Figure 5</a>A.</p><p>(C) Construction of fusion proteins derived from the M22 and M5 proteins. An N-terminal region derived from M22 was fused to the C-terminal part of M5 (residues 104–450 of M5). The fusion proteins contain the Fg-binding B-repeat region of M5.</p><p>(D) Schematic representation of the N-terminal region of different fusion proteins. The sequence of the N-terminal region of M22 is given at the top. Asterisks indicate the position of residues L28, E31, and D40 in M22 (corresponding to the three boxed residues in [B]). The ability of the fusion proteins to bind C4BP, indicated to the right, is based on the results shown in (E).</p><p>(E) Ability of fusion proteins to bind C4BP. The fusion proteins (D) are referred to as M22⁵⁷–M5, etc. Whole-cell lysates of E. coli strains, expressing the indicated proteins from genes carried on pBR322, were analyzed by Western blot using Fg or C4BP as the probe. The strain expressing M5 was used as a negative control. The control blot with Fg showed that the proteins were expressed in <i>E. coli.</i> The presence of double bands probably reflects incomplete processing of signal peptides in E. coli and/or intracellular degradation of M protein in this heterologous host.</p></div