Werner M H, Clore G M, Fisher C L, Fisher R J, Trinh L, Shiloach J, Gronenborn A M
Laboratory of Chemical Physics, NIDDKD, National Institutes of Health, Bethesda, MD 20892-0520, USA.
J Biomol NMR. 1997 Dec;10(4):317-28. doi: 10.1023/a:1018399711996.
The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761-771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456-460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix-turn-helix element and minor groove recognition via protein side-chain intercalation. The presence of intercalation was deduced on the basis of numerous NOEs between several amino acids in the protein and a resonance at 12.33 ppm originally assigned to a DNA imino proton. New NMR experiments now conclusively demonstrate that this resonance, which is located within the DNA imino proton region of the spectrum, arises from the hydroxyl proton of Tyr86. Realization of this error necessitated reanalysis of the intermolecular NOEs. This revealed that the orientation of the ETS1-DBD in the complex is opposite to that originally reported and that a tryptophan residue does not intercalate into the DNA. The calculation of a new ensemble of structures based on the corrected data indicates that the structure of the ETS1-DBD/DNA complex is indeed similar to the X-ray structure of the Pu.1-DBD/DNA complex.
ETS转录因子家族由一组蛋白质组成，这些蛋白质共享一个高度保守的85个氨基酸的DNA结合结构域（DBD）。该家族识别富含嘌呤碱基且具有中心GGAA基序的共有序列。通过核磁共振对已发表的ETS1的DBD/DNA复合物三维结构进行比较[Werner等人（1995年）《细胞》，83卷，761 - 771页]，以及通过X射线晶体学对相关的Pu.1进行比较[Kodandapani等人（1996年）《自然》，380卷，456 - 460页]，发现了一个明显的差异，即蛋白质结构域与其靶序列以相反的极性结合。这个令人惊讶且极不可能的结果促使我们重新审视我们的核磁共振结构。额外的核磁共振实验现在揭示了在最初解释确定ETS1 - DBD在DNA上取向的光谱时存在一个错误。最初报道ETS1 - DBD通过一个双部分基序与DNA结合，该基序涉及通过一个螺旋 - 转角 - 螺旋元件识别大沟以及通过蛋白质侧链插入识别小沟。插入的存在是基于蛋白质中几个氨基酸之间大量的核Overhauser效应（NOE）以及最初归属于一个DNA亚氨基质子的12.33 ppm处的共振推导出来的。新的核磁共振实验现在确凿地证明，这个位于光谱的DNA亚氨基质子区域内的共振来自于Tyr86的羟基质子。意识到这个错误后，有必要重新分析分子间的NOE。这表明复合物中ETS1 - DBD的取向与最初报道的相反，并且一个色氨酸残基不会插入到DNA中。基于校正后的数据计算新的结构集合表明ETS1 - DBD/DNA复合物的结构确实与Pu.1 - DBD/DNA复合物的X射线结构相似。
