Structure of the FHA1 Domain of Yeast Rad53 and Identification of Binding Sites for both FHA1 and its Target Protein Rad9 Hua Liao, Chunhua Yuan, Mei-I Su, Suganya Yongkiettrakul, Dongyan Qin, Hongyuan Li, In-Ja L. Byeon, Dehua Pei* and Ming-Daw Tsai* Departments of Chemistry and Biochemistry, The Ohio State Biochemistry Program, and Campus Chemical Instrument Center, The Ohio State University, Columbus OH 43210, USA Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identi®cation of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 b-strands, which form two large twisted anti-parallel b- sheets folding into a b-sandwich. Three short a-helices were also ident- i®ed. The b-strands are linked by several loops and turns. These struc- tural features of free FHA1 are similar to those of free FHA2, but there are signi®cant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against 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 res- onance analysis showed that a pThr peptide containing this motif, 188 SLEV(pT)EADATFVQ 200 from Rad9, binds to FHA1 with a K d value of 0.36 mM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K d  4-70 mM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously. # 2000 Academic Press Keywords: FHA domain; Rad53; Rad9; phosphopeptide; NMR *Corresponding authors Introduction The forkhead-associated (FHA) domain was ®rst identi®ed as a 55-75 amino acid module found in many proteins from yeast to human, including nuclear protein kinases and transcriptional factors (Hofmann & Bucher, 1995). The conserved regions of FHA domains are not continuous, as three highly This is the third paper in the series about the structure-function relationship of FHA domains. For paper II, see Wang et al. (2000). {The ®rst four authors contributed equally to this work. Abbreviations used: AP, alkaline phosphatase; B, b-alanine; BCIP, 5-bromo-4-chloro-3 0 -indolylphosphate p-toluidine salt; FHA, forkhead-associated domain; GST, glutathione S-transferase; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexa¯uorophosphate; HBS, Hepes buffer saline; HBS-EP, Hepes buffer saline with EDTA and P20; HOBT, 1-hydroxybenzotriazole; HSQC, heteronuclear single-quantum coherence; MALDI-TOF, matrix-assisted laser desorption ionization-time of ¯ight; NHS, N-hydroxysulfosuccinimide; Nle, norleucine; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement correlated spectroscopy; ppm, parts per million; Rad9p, phosphorylated Rad9; TOCSY, total correlation spectroscopy; X, 19 amino acids including norleucine, except cystiene and methionine; Z, norleucine. E-mail addresses of corresponding authors: Pei.3@osu.edu; Tsai.7@osu.edu doi:10.1006/jmbi.2000.4291 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 304, 941±951 0022-2836/00/050941±11 $35.00/0 # 2000 Academic Press