THREE-DIMENSIONAL STRUCTURES OF BIOLOGICAL MACROMOLECULES

研究了NK-2类同源结构域的野生型和突变体与罕见的18碱基对dna一致片段的三维结构。针对这些单位点突变的相应转基因实验已经或正在进行中。已经确定了核苷酸序列特异性结合的相互作用。对同源域的7、35、41、52、54和56个位置进行突变分析。从酪氨酸到蛋氨酸的第54位突变导致同位结构域对DNA一致序列的结合亲和力降低一个数量级。令人惊讶的是，在54号位置有这种突变并且内源性nk -2基因被消除的转基因果蝇系并不致命。位置35从丙氨酸到苏氨酸的突变导致同源结构域在-5摄氏度的温度下无法在溶液中采用折叠构象。这种突变确实会导致胚胎死亡。丙氨酸转苏氨酸突变体同源结构域与vnd/NK-2靶DNA序列特异性结合，但其亲和力比野生型同源结构域低50倍。虽然突变体在DNA结合状态下的三维结构具有三个螺旋，并且显示出与野生型同源域相似的螺旋-转-螺旋DNA结合基序的特征，但在突变体类似物中观察到明显的结构扭曲。野生型同位结构域与残基35的主酰胺氧形成不寻常的i， i-5氢键。在突变体中，这个酰胺质子氢键被改变，蛋白质在螺旋- ii区域的结构相对于野生型类似物是扭曲的。初步结论是，整体DNA结合亲和力较低的突变导致胚胎致死性，而选择不同启动子位点的突变可能导致表型改变，但不一定致命。-核磁共振vnd/NK-2同源结构域果蝇三维结构位点突变

The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class bound to an uncommon 18 base-pairDNA consensus segment have been studied. Corresponding transgenic experiments on these single site mutations have been or are being carried out. The interactions responsible for the nucleotide sequence- specific binding have been identified. Mutation analysis was carried out on for positions (7, 35, 41,52, 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. Surprisingly, transgenic Drosophila lines with this mutation in position 54 and with the endogenous NK-2genes eliminated are not lethal. 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. This mutation does result in embryonic lethality. 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 has three helices and shows the characteristic helix-turn-helix DNA binding motif similar to that of the wild-type homeodomain, a notable structural distortion in the mutant analog is observed. 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. The tentative conclusion is that mutations that globally lower DNA binding affinity lead to embryonic lethality, whereas mutations that select for a different set of promotor sites likely result in altered phenotypes but are not necessarily lethal. - NMR vnd/NK-2 homeodomain Drosophila three- dimensional structure site mutation