Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9

Forkhead-associated (FHA) domains have been shown to recognize both pThr an d pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from S accharomyces cerevisiae, and its complex with a pTyr peptide, have been rep orted recently. We now report the solution structure of the other FHA domai n of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta -s trands, which form two large twisted anti-parallel beta -sheets folding int o a beta -sandwich. Three short alpha -helices were also identified. The be ta -strands are linked by several loops and turns. These structural feature s of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] agai nst FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed th at a pThr peptide containing this motif, (SLEV)-S-188(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K-d value of 0.36 muM Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K-d = 4-70 muM). The se results suggest that Thr192 of Rad9 is the likely phosphorylation site r ecognized by the FHA1 domain of Rad53. The tight-binding peptide was then u sed to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 do main and a pTyr peptide derived from Rad9 reported previously. (C) 2000 Aca demic Press.