Molecular structure and specific interaction of fha domains of saccharomyces cerevisiae rad53

by Yongkiettrakul, Suganya

Abstract (Summary)
Combinatorial phosphopeptide library screening and biochemical approaches were used to identify and analyze specific binding motifs of FHA1 and FHA2 domains of yeast Rad53. The results of pThr-peptide library screening indicate the absolute requirement of Asp at the +3 position of phosphothreonine peptide (pTXXD) for the FHA1 domain and the preference of Ile/Leu at the +3 position (pTXXI/L) for the FHA2 domain. As a result of probing these specific-binding motifs on Rad9, tight binding Rad9-derived pThr peptides of the FHA1 and FHA2 domain have been identified: 188SLEV(pT)EADATFVQ200 and 599EVEL(pT)QELP607, respectively. NMR-based structural determination of both FHA complexes with their tight-binding pThr peptides has elucidated molecular interactions between the FHA proteins and pThr peptides. The structural basis of ligand binding specificity of the complexes could suggest that the specific recognition at +3Asp of the FHA1 domain is determined by a salt-bridge interaction of Arg83, whereas the FHA2 domain specifically recognizes +3Ile/Leu by a hydrophobic interaction of Ile681. On the basis of molecular interactions of Rad53 FHA domains binding to pThr peptides, non-conserved residues Gly133 and Gly135 of the FHA1 domain and Ile681 and Asp683 of the FHA2 domain, which are located outside of the core FHA region of yeast Rad53 FHA domains, are proposed to contribute to the specific recognition of the +3 position of different phosphopeptides. By rational mutagenesis and binding studies, the results have confirmed a significant contribution of these residues in the difference ligand-binding specificity between FHA1 and FHA2 domains. In addition, results of structural analyses by NMR indicate that some of these residues are also important for the structural integrity of the peptide binding loops. Furthermore, on the basis of results obtained from the in vitro studies, yeast genetic approaches were used to identify biological binding sites of the Rad53 FHA domains. Preliminary results suggest that Thr560, Thr603, Tyr829, and Thr933 of the Rad9 are likely involved in the Rad9 function and both Thr603 and Tyr829 are potential phosphorylation sites of the Rad9, recognized by the FHA2 domain. In addition to Rad9, the results suggest that the FHA2 domain may have an additional binding partner.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:fha domain rad53 rad9 phosphopeptide nmr


Date of Publication:01/01/2004

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