The objective of this proposal is to characterize the structure and specificity of a new class of phosphoprotein binding domain, the """"""""forkhead-associated domain"""""""" (FHA domain), from four important proteins: human Chk2, human Nibrin, human Ki67, and yeast Rad53. All these proteins (except Ki67) are involved in signaling events in the DNA damage response, and the functions of the three human proteins are directly related to cancer. The project consists of five specific aims and involves two collaborators (L-J. Byeon and D. Pei).
Specific Aim 1 is to express different FHA domains and determine their tertiary structures by NMR.
Specific Aim 2 is to perform rigorous analyses of the ligand specificity of the FHA domains by use of combinatorial peptide libraries containing pTyr, pSer, or pThr, in combination with physical methods (mass spectrometry, surface plasmon resonance, fluorescence resonance energy transfer, and NMR).
Specific Aim 3 is to determine the structures of the complexes of the FHA domain with the tight-binding phosphopeptides identified in the previous specific aim by NMR.
Specific Aim 4 is to perform quantitative analyses of the structure-function relationship of FHA domains. On the basis of the structures of the complex, the principal investigator will mutate key recognition residues in the FHA domain and determine the changes in the binding affinity. Furthermore, he will try to engineer new phosphopeptide specificity. The goal is to fully understand the quantitative structure-function relationship for different FHA domains.
Specific Aim 5 is to identify the natural FHA-binding site of Rad9 on the basis of the optimal peptide sequences identified in Aim 2. A combination of site-directed mutagenesis, binding assays, and peptide mapping will be employed. Overall, the information generated will lead to an understanding of the chemical mechanism of PHA domains in cellular controls related to DNA damage and cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA087031-02
Application #
6514624
Study Section
Biochemistry Study Section (BIO)
Program Officer
Gallahan, Daniel L
Project Start
2001-03-05
Project End
2005-02-28
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
2
Fiscal Year
2002
Total Cost
$232,115
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Mahajan, Anjali; Yuan, Chunhua; Lee, Hyun et al. (2008) Structure and function of the phosphothreonine-specific FHA domain. Sci Signal 1:re12
Byeon, In-Ja L; Li, Hongyuan; Song, Haiyan et al. (2005) Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67. Nat Struct Mol Biol 12:987-93
Mahajan, Anjali; Yuan, Chunhua; Pike, Brietta L et al. (2005) FHA domain-ligand interactions: importance of integrating chemical and biological approaches. J Am Chem Soc 127:14572-3
Pike, Brietta L; Yongkiettrakul, Suganya; Tsai, Ming-Daw et al. (2004) Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae. Mol Cell Biol 24:2779-88
Li, Hongyuan; Byeon, In Ja L; Ju, Yong et al. (2004) Structure of human Ki67 FHA domain and its binding to a phosphoprotein fragment from hNIFK reveal unique recognition sites and new views to the structural basis of FHA domain functions. J Mol Biol 335:371-81
Yongkiettrakul, Suganya; Byeon, In-Ja L; Tsai, Ming-Daw (2004) The ligand specificity of yeast Rad53 FHA domains at the +3 position is determined by nonconserved residues. Biochemistry 43:3862-9
Pike, Brietta L; Yongkiettrakul, Suganya; Tsai, Ming-Daw et al. (2003) Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation. J Biol Chem 278:30421-4
Qin, Dongyan; Lee, Hyun; Yuan, Chunhua et al. (2003) Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies. Biochem Biophys Res Commun 311:803-8
Byeon, I J; Yongkiettrakul, S; Tsai, M D (2001) Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes. J Mol Biol 314:577-88
Yuan, C; Yongkiettrakul, S; Byeon, I J et al. (2001) Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53. J Mol Biol 314:563-75