PROTEIN DNA CONTACTS IN THE STRUCTURE OF A HOMEODOMAIN DNA COMPLEX DETERMINED BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY IN SOLUTION
PROTEIN DNA CONTACTS IN THE STRUCTURE OF A HOMEODOMAIN DNA COMPLEX DETERMINED BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY IN SOLUTION
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DOI：
10.1002/j.1460-2075.1990.tb07505.x
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发表时间：
1990-10-01
期刊：
影响因子：
11.4
通讯作者：
WUTHRICH, K
中科院分区：
文献类型：
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作者：
OTTING, G;QIAN, YQ;WUTHRICH, K
突变体ANTP(C39.fwdarw.)1：1复合体S)的同源结构域，在水溶液中用核磁共振波谱研究了BS2结合位点对应的14bpDNA片段。该化合物的相对分子质量为17800，与核磁共振化学位移时间标度相比，其寿命较长。对三维结构的研究是基于使用全15N标记的蛋白质、带有15N(ω2)半滤膜的二维同核质子NOESY和异核三维核磁共振实验。根据几乎完全的序列特异性共振指定，蛋白质和DNA在自由态和复合体中都有相似的构象。可以确定足够数量的分子间1H-1H Overhauser效应(NOE)以实现蛋白质与DNA的独特对接，这是使用椭球体算法实现的。在该复合体中，在同源结构域的螺旋-转角-螺旋模体中延长的第二螺旋与DNA的主沟之间存在分子间的NOE。NOE与DNA的其他接触涉及紧接在螺旋-转弯-螺旋片段之前的多肽环和Arg5。后一种接触是特别有趣的，这既是因为Arg5到达小槽，也是因为在游离的ANTP(C39.fwdarw.S)同源域中，没有找到明显灵活的N-末端片段的定义空间结构，该片段包含残基0-6。
The 1:1 complex of the mutant Antp (C39.fwdarw. S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17 800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(.omega.2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39.fwdarw.S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.