THREE-DIMENSIONAL STRUCTURES OF BIOLOGICAL MACROMOLECULES

vnd/NK-2的三维结构 与罕见18碱基对DNA结合的同源结构域 分析了含有5 '-CAAGTG-3'的共有片段 通过核磁共振光谱。这些相互作用导致了 已经鉴定了核苷酸序列特异性结合，其中 54位的酪氨酸以前被证明是主要的 不寻常的共有核苷酸序列的决定因素。 对位置（35、52、54和55）进行突变分析。 56)在homeodomain。54位从酪氨酸突变为 甲硫氨酸的结果，在一个数量级的减少， 同源结构域对DNA共有区的结合亲和力 顺序位置35从丙氨酸突变为苏氨酸， 在不能采取折叠构象的同源结构域中 在-5摄氏度以下的溶液中是自由的。的 丙氨酸到苏氨酸突变的同源域特异性地结合到 vnd/NK-2靶DNA序列，但具有50倍的亲和力 低于野生型同源结构域。虽然 DNA结合状态下突变体的三维结构 显示出与所述的螺旋-转角-螺旋特性类似的特征性螺旋-转角-螺旋特性。 野生型同源结构域，一个显着的结构偏差，在 观察到亮氨酸-40的酰胺质子的突变类似物。的 野生型同源结构域形成不寻常的I，I-5氢键， 残基35的骨架酰胺氧。在突变体中， 质子氢键被改变， 螺旋-II区域相对于野生型的螺旋-II区域是扭曲的， analog. .

The three-dimensional structure of the vnd/NK-2 homeodomain bound to an uncommon 18 base-pair DNA consensus segment that contains 5'-CAAGTG-3' has been analyzed by NMR spectroscopy. The interactions responsible for the nucleotide sequence-specific binding have been identified, where tyrosine in position 54 previously was shown to be the major determinant of the unusual consensus nucleotide sequence. Mutation analysis was carried out on for positions (35, 52, 54, and 56) in the homeodomain. Mutation of position 54 from tyrosine to methionine results in a decrease of one order of magnitude in the binding affinity of the homeodomain for the DNA consensus sequence. Mutation of position 35 from alanine to threonine results in a homeodomain that is unable to adopt a folded conformation free in solution at temperatures down to -5 degrees Celsius. The alanine to threonine mutant homeodomain specifically binds to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower that that of the wild-type homeodomain. Although the three-dimensional structure of the mutant in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i, i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the mutant this amide proton hydrogen bond is altered and the structure of the protein in the region of helix-II is distorted relative to that of the wild-type analog. .