Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53.
Rad53, a yeast checkpoint protein involved in regulating the repair of DNA damage, contains two forkhead-associated domains, FHA1 and FHA2. Previous combinatorial library screening has shown that FHA1 strongly selects peptides containing a pTXXD motif. Subsequent location of this motif within the sequence of Rad9, the target protein, coupled with spectroscopic analysis has led to identification of a tight binding sequence that is likely the binding site of FHA1: (188)SLEV(pT)EADATFVQ(200). We present solution structures of FHA1 in complex with this pT-peptide and with another Rad9-derived pT-peptide that has ca 30-fold lower affinity, (148)KKMTFQ(pT)PTDPLE(160). Both complexes showed intermolecular NOEs predominantly between three peptide residues (pT, +1, and +2 residues) and five FHA1 residues (S82, R83, S85, T106, and N107). Furthermore, the following interactions were implicated on the basis of chemical shift perturbations and structural analysis: the phosphate group of the pT residue with the side-chain amide group of N86 and the guanidino group of R70, and the carboxylate group of Asp (at the +3 position) with the guanidino group of R83. The generated structures revealed a similar binding mode adopted by these two peptides, suggesting that pT and the +3 residue Asp are the major contributors to binding affinity and specificity, while +1 and +2 residues could provide additional fine-tuning. It was also shown that FHA1 does not bind to the corresponding pS-peptides or a related pY-peptide. We suggest that differentiation between pT and pS-peptides by FHA1 can be attributed to hydrophobic interactions between the methyl group of the pT residue and the aliphatic protons of R83, S85, and T106 from FHA1.
Yuan
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Department of Chemistry, The Ohio State University, Columbus OH 43210, USA.
Yongkiettrakul
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Byeon
I J
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Zhou
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Tsai
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MD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D064447:Checkpoint Kinase 2; D018360:Crystallography, X-Ray; D051858:Forkhead Transcription Factors; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry*
D018797:Cell Cycle Proteins; D051858:Forkhead Transcription Factors; D008024:Ligands; D009687:Nuclear Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; C069930:rad9 protein; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae