ELONGATION OF HELIX-III OF THE NK-2 HOMEODOMAIN UPON BINDING TO DNA - A SECONDARY STRUCTURE STUDY BY NMR
ELONGATION OF HELIX-III OF THE NK-2 HOMEODOMAIN UPON BINDING TO DNA - A SECONDARY STRUCTURE STUDY BY NMR
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DOI：
10.1021/bi00254a014
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发表时间：
1994-12-20
期刊：
影响因子：
2.9
通讯作者：
FERRETTI, JA
中科院分区：
文献类型：
--
作者：
TSAO, DHH;GRUSCHUS, JM;FERRETTI, JA
用二维和三维（2D和3D）核磁共振波谱技术测定了黑腹果蝇NK-2基因编码的同源结构域在游离和dna结合状态下的二级结构。用质子和N-15对大肠杆菌合成的含有同源结构域的77个氨基酸残基蛋白进行了研究。根据NOE连通性、邻域偶联常数和质子-氘交换行为，发现在没有DNA的情况下，蛋白质的三个螺旋片段由同位结构域氨基酸残基10-22、28-38和42-52组成。自由NK-2和其他同源结构域之间的主要结构差异是第二螺旋的内部流动性增加，第三螺旋的长度较短，也称为识别螺旋。尽管有较短的螺旋结构，与其他同源结构域相比，NK-2显示出与DNA的高亲和力结合(k(D) = 2.0 x 10(-10) M；L.-H。Wang和M. Nirenberg，未发表的结果)。NK-2与双链DNA复合物（TGTGTCAAGTGGCTGT）的形成显著提高了蛋白的热稳定性。T-m从25℃（游离NK-2）增加到47℃（dna结合NK-2）。此外，还观察到螺旋m长度的急剧增加。在没有DNA的情况下，DNA识别螺旋的长度为11个氨基酸残基（42-52个残基），而在存在DNA的情况下，该螺旋的长度延伸至19个氨基酸（42-60个残基）。在质子-氘交换实验中，与游离NK-2同源结构域相比，dna结合蛋白的酰胺质子交换速率一般较慢，这表明NK-2具有稳定的构象。提出了一种非结合NK-2三级结构的初步模型。
The secondary structure of the homeodomain encoded by the NK-2 gene from Drosophila melanogaster, in both the free and DNA-bound states, was determined in solution using two- and three-dimensional (2D and 3D) NMR Spectroscopy. Proton and N-15 studies were carried out on a 77 amino acid residue protein that contains the homeodomain, which was synthesized in Escherichia coli. On the basis of NOE connectivities, vicinal coupling constants, and proton-deuterium exchange behavior, three helical segments were found that consist of homeodomain amino acid residues 10-22, 28-38, and 42-52 for the protein in the absence of DNA. The major structural differences between free NK-2 and other homeodomains are the increased internal mobility of the second helix and the shorter length of the third helix, also termed the recognition helix. Despite this shorter helix, NK-2 exhibits high-affinity binding to DNA compared to other homeodomains (k(D) = 2.0 x 10(-10) M; L.-H. Wang and M. Nirenberg, unpublished results). The formation of the complex of NK-2 with the duplex DNA (TGTGTCAAGTGGCTGT) significantly increases the thermal stability of the protein. The T-m increases from 25 degrees C (free NK-2) to >47 degrees C (DNA-bound NK-2). Also, a dramatic increase in the length of helix m is observed. In the absence of DNA, the DNA recognition helix is 11 amino acid residues long (residues 42-52), whereas in the presence of DNA, the length of this helix extends to 19 amino acids (residues 42-60). The exchange rates of the amide protons in the proton-deuterium exchange experiments are slower in general for the DNA-bound protein than for the free NK-2 homeodomain, which is indicative of a stable conformation for NK-2. A preliminary model of the unbound NK-2 tertiary structure is presented.