The first site is partially BAY-1895344 conserved and formed by His240, Arg248 and Arg264, while the SGC2085 second site is completely conserved and formed by Lys173 from L1�C2, Ser268, Thr269 and Arg270 from L10�C11, and Glu202 and His203 from L4�C5. Notably, residues of the second site are arranged in a similar fashion to the canonical pThr binding site residues. These additional binding sites could indicate an extended recognition surface for anionic groups of potential ligands. Recognition of more than one phosphorylation sites by an FHA domain, such as observed in the FHA domain of Dun1, can significantly increase the binding affinity. The potential, second binding site of kanadaptin-FHA is located on the opposite side compared to Dun1-FHA, with respect to the common, conserved pThr-binding sites. The structure of the FHA domain of kanadaptin is very similar to that of other FHA domains. For example, it aligns with the Arabidopsis thaliana Dawdle FHA domain with an RMSD of 1.8 A ? over 120 Ca atom pairs and a sequence identity of 33%. The putative pThr recognition site is also very similar to that of other FHA domains, in particular the Dawdle FHA domain. One residue of particular interest in the pThr binding region is His240 that is strictly conserved in all FHA domains of kanadaptin orthologs, but is not conserved across other non-kanadaptin FHA domains where Asn is instead found at the equivalent position. However, in both kanadaptin FHA and non-kanadaptin FHA domains, the side chain at this position hydrogen bonds with the backbone carbonyl group of the amino acid immediately following the pThr amino acid. Therefore, this structurally conserved residue functions in anchoring the target peptide within the recognition site and may also help define the preferred residue at pThr+1. This is consistent with other studies that have explored the side chain specificities of the pThr binding site. For example, it has previously been shown that peptide specificity is modulated by the chemical nature of the side chains at positions pThr+3, +1, 22 and 23. Overall, peptides bound to FHA domains share a comparable conformation.