II. Structure and Specificity of the Interaction between the FHA2 Domain of Rad53 and Phosphotyrosyl Peptides{ Peng Wang, In-Ja L. Byeon*, Hua Liao, Kirk D. Beebe Suganya Yongkiettrakul, 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 The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the ®rst structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr- containing peptide 826 EDI(pY)YLD 832 from Rad9, with a moderate af®nity (K d ca. 100 mM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phos- phate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the 2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence speci- ®city of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes speci®c sequences C-term- inal to pTyr with the following consensus: XX(pY)N 1 N 2 N 3 , where N 1  Leu, Met, Phe, or Ile, N 2  Tyr, Phe, Leu, or Met, and N 3  Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FY- MIR, bind FHA2 with K d values of 1.1 and 5.0 mM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences. # 2000 Academic Press Keywords: FHA domain; Rad53; phosphotyrsine; phosphopeptide *Corresponding authors Introduction Protein-protein interaction is one of the essential mechanisms used in cellular signaling processes. Interactions between two proteins are often mediated by small modular domains, which are autonomously folded and recognize short stretches of amino acid residues in their partner proteins. A number of such modules, including SH2, SH3, PTB, and 14-3-3 domains, have been identi®ed in signaling proteins of organisms from yeast to human (Pawson & Scott, 1997). Recently, a new module, the forkhead-associated (FHA) domain, has been identi®ed by sequence analyses (Hofmann & Bucher, 1995). It was ®rst identi®ed within a subset of forkhead transcriptional factors, located outside of the conserved DNA-binding forkhead domain. It was subsequently found in more than 20 other proteins, mostly nuclear pro- tein kinases and transcriptional factors. New FHA- containing proteins of diverse functions continue to be identi®ed. More recent additions include HuCdsl (or Chk2) from humans (Brown et al., 1999; Matsuoka et al., 1998) and Dmnk from Droso- phila melanogaster (Oishi et al., 1998). Both proteins participate in the cellular response to DNA damage. Recently, an FHA domain was identi®ed {Paper I in this series is Liao et al. (1999). Abbreviations used: B, b-alanine; EDTA, ethylenediamine tetraacetic acid; FHA, forkhead- associated domain; FRET, ¯uorescence energy transfer; GST, glutathione S-transferase; HBTU, 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa¯uorophosphate; HOBT, 1-hydroxybenzotriazole; HSQC, heteronuclear single-quantum coherence; MALDI, matrix-assisted laser desorption ionization; MS, mass spectroscopy; Nle, norleucine; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement correlated spectroscopy; PTB domain, phosphoprotein-binding domain. E-mail addresses of the corresponding authors: Byeon.2@osu.edu, Pei.3@osu.edu, Tsai.7@osu.edu doi:10.1006/jmbi.2000.4095 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 302, 927±940 0022-2836/00/040927±14 $35.00/0 # 2000 Academic Press