SmMBD2/3 contains amino acid residues critical for binding to 5mC templates.

<p><b>(A)</b> Diagrammatic representation of the 314 amino acid SmMBD2/3 (encoded by Smp_138180) illustrating the methyl-CpG (mCpG) binding domain (PF01429), two putative predicted bipartite nuclear localisation signals (NLS), a coiled-coil domain (CC) and a C-terminal domain of methyl CpG binding protein 2 and 3 (PF140489). Amino acid positions are indicated in bold numbering. <b>(B)</b> A multiple sequence alignment of the methyl-CpG (mCpG) binding domains (PF01429) collected from SmMBD2/3 (italics and contained in a blue box) and MBD homologs was generated. MBDs unable to bind 5mC are indicated in red. Highly conserved residues are highlighted in turquoise and moderately conserved residues are shaded grey. A ‘*’ indicates amino acid residues that contribute to 5mC binding as assessed by mutational studies (summarised in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.t001" target="_blank">Table 1</a>). A ‘#’ signifies additional amino acid residues that directly interact with 5mC and a ‘:’ indicates amino acid residues that interact with the DNA phosphate backbone [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref021" target="_blank">21</a>]. Amino acid insertions in AmMBD1 and DmMBD2/3 are indicated in black boxes above and below the alignment, respectively. AmMBD1: XP_003250634.1, HsMBD1: NP_002375.1, MmMBD1: NP_038622.2, HsMeCP2: NP_001104262.1, MmMeCP2: NP_001075448.1, XlMeCP2: NP_001081854.1, HsMBD4: NP_001263201.1, MmMBD4: NP_034904.2, HsMBD2: NP_003918.1, MmMBD2 NP_034903.2, GgMBD2: NP_001012403.1, HsMBD3: NP_001268382.1, MmMBD3: NP_038623.1, XlMBD3: AAD55389.1, BmMBD2/3: XP_004929675.1, ApMBD2/3: ACF05483.1, HpMBD2/3: ACF05485.1, AdMBD2/3: [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref025" target="_blank">25</a>], SmMBD2/3: CCD59176.1, DmMBD2/3: NP_731370.1. <b>(C)</b> Ribbon representation of SmMBD2/3 homology model (green) depicts 5mC-interacting residues (stick representation: amino acids in red, 5mC in orange) found within PF01429. The SmMD2/3 model was generated using the solved crystal structure of <i>G</i>. <i>gallus</i> MBD2 co-complexed with methylated DNA as detailed in the <i>Materials and Methods</i>. The tetra-amino acid archetypal binding pocket is indicated (red).