Schott O, Billeter M, Leiting B, Wider G, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zurich, Switerland.
J Mol Biol. 1997 Apr 4;267(3):673-83. doi: 10.1006/jmbi.1997.0905.
The nuclear magnetic resonance (NMR) solution structure of the non-classical homeodomain from the rat liver LFB1/HNF1 transcription factor was determined with the program DIANA from an input of 1356 nuclear Overhauser enhancement (NOE) upper distance constraints and 228 dihedral angle constraints collected using experiments with the unlabelled, the uniformly 15N-labelled and the uniformly 13C-labelled protein. Out of a group of 50 independently calculated conformers the 20 conformers with the smallest residual DIANA target function values were refined by energy minimization with the program OPAL and are used to represent the NMR structure. The average of the pairwise root-mean-square deviations (r.m.s.d.) of these 20 individual NMR conformers relative to the mean coordinates is 0.73 A (1 A = 0.1 nm) for the backbone atoms N, C(alpha) and C' of residues 15 to 82. The chain-terminal polypeptide segments 1-14 and 90-99 are disordered in solution. The globular fold contains three well-defined helices comprising the residues 19 to 29, 37 to 53 and 71 to 81, and the third helix is extended by a less well-ordered fourth helix with residues 82 to 89, which coincides with corresponding observations in "classical" homeodomains. Side-chain analysis resulted in 33 "best-defined" side-chains, with global displacements smaller than 1.1 A, and addition of these side-chains to the global superposition of residues 15 to 82 resulted in a r.m.s.d of 0.81 A. The protein contains two hydrophobic cores, one of which corresponds to the helical packing seen in classical homeodomains, while the other one stabilizes the conformation of the 21-residue insertion between helices II and III. The individual helices and their relative spatial arrangements are stabilized by a variety of structural motifs, which include medium-range and long-range hydrogen bonds and salt bridges. Detailed comparison with the Antennapedia homeodomain, and studies of the complex formation with an operator DNA half-site provided initial information on the DNA-binding mode of the LFB1/HNF1 homeodomain.
利用DIANA程序，根据1356个核Overhauser效应（NOE）上限距离约束和228个二面角约束确定了大鼠肝脏LFB1/HNF1转录因子非经典同源结构域的核磁共振（NMR）溶液结构，这些约束是通过对未标记、均匀15N标记和均匀13C标记的蛋白质进行实验收集得到的。在一组50个独立计算的构象中，通过OPAL程序进行能量最小化，对20个具有最小残余DIANA目标函数值的构象进行了优化，并用于表示NMR结构。对于15至82位残基的主链原子N、C(α)和C'，这20个单独的NMR构象相对于平均坐标的成对均方根偏差（r.m.s.d.）平均值为0.73 Å（(1 Å = 0.1 nm）。链端多肽片段1 - 14和90 - 99在溶液中无序。球状折叠包含三个明确的螺旋，分别由19至29、37至53和71至81位残基组成，第三个螺旋由82至89位残基构成的不太有序的第四个螺旋延伸，这与“经典”同源结构域中的相应观察结果一致。侧链分析得到33个“定义最清晰”的侧链，其全局位移小于1. A，将这些侧链添加到15至82位残基的全局叠加中，得到的r.m.s.d为0.81 Å。该蛋白质包含两个疏水核心，其中一个对应于经典同源结构域中所见的螺旋堆积，而另一个则稳定了螺旋II和III之间21个残基插入片段的构象。各个螺旋及其相对空间排列通过多种结构基序得以稳定，这些结构基序包括中程和远程氢键以及盐桥。与触角足同源结构域的详细比较以及与操纵子DNA半位点形成复合物的研究，为LFB1/HNF1同源结构域的DNA结合模式提供了初步信息。
