The molecular basis of FHA Domain : Phosphopeptide binding specificity andimplications for phospho-dependent signaling mechanisms

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modul es that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, i ncluding those within a number of DNA damage checkpoint kinases, and determ ined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonin e peptide, at 1.6 Angstrom resolution. The structure reveals a striking sim ilarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain comple xes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in proka ryotes and eukaryotes.