Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53

The forkhead-associated (FHA) domain is a 55-75 amino acid residue module f ound in >20 proteins from yeast to human. It has been suggested to particip ate in signal transduction pathways, perhaps via protein-protein interactio ns involving recognition of phosphopeptides. Neither the structure nor the ligand of FHA is known. Yeast Rad53, a checkpoint protein Involved in DNA d amage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 ( residues 601-664), the second of which recognizes phosphorylated Rad9. We h erein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730). The structure consists of a beta-sandwich containing t wo antiparallel beta-sheets and a short, C-terminal alpha-helix. Binding ex periments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site in volves residues highly conserved across FHA domains. The results, along wit h other recent reports, suggest that FHA domains could have pTyr and pSer/T hr dual specificity. (C) 1999 Academic Press.