Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53.
Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53.
复制标题
两种 FHA1-磷酸苏氨酸肽复合物的溶液结构提供了对酵母 Rad53 的 FHA1 配体特异性的结构基础的深入了解。
DOI：
10.1006/jmbi.2001.5140
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发表时间：
2001
影响因子：
5.6
通讯作者：
Tsai,MD
中科院分区：
文献类型：
--
作者：
Yuan,C;Yongkiettrakul,S;Byeon,IJ;Zhou,S;Tsai,MD
Rad 53是一种参与调节DNA损伤修复的酵母检查点蛋白，含有两个叉头相关结构域FHA 1和FHA 2。先前的组合文库筛选已经显示FHA 1强烈选择含有pTXXD基序的肽。随后在Rad 9（靶蛋白）序列中定位该基序，结合光谱分析，鉴定出一个紧密结合序列，可能是FHA 1：188 SLEV（pT）EADATFVQ 200的结合位点。我们呈现了FHA 1与该pT肽以及另一种Rad 9衍生的pT肽（亲和力低约30倍）148 KKMTFQ（pT）PTDPLE 160的复合物的溶液结构。两种复合物均显示分子间NOE主要在3个肽残基（pT、+1和+2残基）和5个FHA 1残基（S82、R83、S85、T106和N107）之间。此外，在化学位移扰动和结构分析的基础上，涉及以下相互作用：pT残基的磷酸基团与N86的侧链酰胺基团和R70的胍基，以及Asp的羧酸基团（在+3位）与R83的胍基。生成的结构揭示了这两种肽采用的相似结合模式，表明pT和+3残基Asp是结合亲和力和特异性的主要贡献者，而+1和+2残基可以提供额外的微调。还显示FHA 1不与相应的pS-肽或相关的pY-肽结合。我们认为，由FHA 1的pT和pS-肽之间的分化可以归因于从FHA 1的pT残基的甲基和R83，S85，和T106的脂肪族质子之间的疏水相互作用。
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 FHA1:188SLEV(pT)EADATFVQ200. 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,148KKMTFQ(pT)PTDPLE160. 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.