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 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 domai...

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Bibliographic Details
Published in:Journal of molecular biology 2000-12, Vol.304 (5), p.941-951
Main Authors: Liao, Hua, Yuan, Chunhua, Su, Mei-I, Yongkiettrakul, Suganya, Qin, Dongyan, Li, Hongyuan, Byeon, In-Ja L., Pei, Dehua, Tsai, Ming-Daw
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Binding Sites
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - metabolism
Checkpoint Kinase 2
FHA domain
Forkhead Transcription Factors
Ligands
Models, Molecular
Molecular Sequence Data
NMR
Nuclear Magnetic Resonance, Biomolecular
Nuclear Proteins - chemistry
Nuclear Proteins - metabolism
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Peptide Library
phosphopeptide
Phosphopeptides - metabolism
Phosphorylation
Phosphothreonine - metabolism
Protein Kinases - chemistry
Protein Kinases - metabolism
Protein Structure, Secondary
Protein Structure, Tertiary
Protein-Serine-Threonine Kinases
Rad53
Rad53 protein
Rad9
Rad9 protein
Saccharomyces cerevisiae
Saccharomyces cerevisiae - chemistry
Saccharomyces cerevisiae Proteins
Sequence Alignment
Substrate Specificity
Surface Plasmon Resonance
Thermodynamics
Transcription Factors - chemistry
Transcription Factors - metabolism
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Summary: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 identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 β-strands, which form two large twisted anti-parallel β-sheets folding into a β-sandwich. Three short α-helices were also identified. The β-strands are linked by several loops and turns. These structural features 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] 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 192TEAD195 in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, 188SLEV(pT)EADATFVQ200 from Rad9, binds to FHA1 with a Kd value of 0.36μM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (Kd=4-70μM). 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.
ISSN:0022-2836
1089-8638
DOI:10.1006/jmbi.2000.4291