Three-dimensional Structures Of Biological Macromolecule

已经研究了NK-2类中同源结构域的野生型和突变体的三维结构，特别是vnd/NK-2同源结构域，以及全长CSX/NKX-2.5和NKX-3.1蛋白质，两者都处于游离状态和与DNA结合。还在游离和DNA结合状态下研究了含有vnd/NK-2同源结构域、接头和NK-2特异性结构域的额外构建体。后一项研究表明，只有同源结构域本身直接与DNA相互作用。我们的转基因研究表明，Y 54 M的改变作为一个敲除，下调ind和msh的能力显然是完全抑制。酸性结构域缺失突变体表现出有限的下调能力。相比之下，tinman和NK-2盒缺失突变体在抑制ind和msh的能力方面都表现为功能性vnd/NK-2基因。同源结构域中的另一个单个氨基酸残基突变H52 R在结构和热方面稳定了同源结构域。然而，在转基因实验中的这种突变对胚胎是有毒的，并且一旦mRNA的表达开始，就完全阻止了生长。将在体外游离和结合至DNA的Y 54 M vnd/NK-2同源结构域的NMR测定的三级结构与野生型类似物进行比较。观察到的突变体同源结构域的唯一结构差异是在与DNA的复合物中，并且涉及甲硫氨酸与DNA的A2而不是C3的更紧密的相互作用。同源结构域与DNA相互作用的这种变化导致同源结构域-DNA复合物的寿命的显著改变。在四个系统中研究了寿命; 1）与其同源DNA结合的野生型同源结构域，2）与含有5 ′- CAATGG -3 ′的DNA序列结合的野生型同源结构域，所述5 ′- CAATGG -3 ′是在位置54含有甲硫氨酸的同源结构域的共有序列，3）与野生型核心DNA共有序列结合的Y 54 M同源结构域，和4）与含有5 ′- CAATGG 3 ′作为其核心的DNA结合的Y 54 M同源结构域。与野生型核心DNA共有序列结合的野生型同源结构域的寿命（即，#1）至少比可能性2 - 4长一个数量级。这里的含义或假设是，转录激活复合物的寿命必须足够长，以确保适当的胚胎发育。为了进一步研究这一假设，使用vnd/NK-2基因5'上游区的几个750碱基对序列作为启动子进行CAT和β-半乳糖苷酶报告基因测定。初步结果表明，5' - CAAGTG - 3'或5' - CAATGG - 3' DNA片段的改变显著改变了CAT和β-半乳糖苷酶的表达。激活和抑制都取决于所做的具体变化。由单个氨基酸残基置换引起的这些变化构成了胚胎发生过程中Y 54 M vnd/NK-2基因异位表达后观察到的表型改变的分子基础。NK-2类同源异型盒基因在发育中的调节作用和高度明确的同源异型域-DNA相互作用之间的重要关系由此建立。这些研究也证明了序列和结构模块性与功能模块性之间的关系，在上面的例子中，只有同源结构域似乎是下调ind和msh所必需的。对与房间隔缺损相关的突变CSX/NKX-2.5同源域的研究表明，Y 54 C突变蛋白的三级结构没有改变，但与功能性DNA结合位点的相互作用被修饰。Y 54 C CSX/NKX-2.5蛋白是完全无功能的。这种蛋白质与DNA相互作用的改变可能构成了疾病的分子基础。最近的结构研究表明，在不与DNA或其他蛋白质因子结合的情况下，CSX/NKX-2.5蛋白质中只有一部分是同源结构域本身。

The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class, specifically the vnd/NK-2 homeodomain, and the full length CSX/NKX-2.5, and NKX-3.1 proteins, both in the free state and bound to DNA have been investigated. Ad additional construct that contains both the vnd/NK-2 homeodomain, a linker and the NK-2 specific domain also were studied in the free and DNA bound states. This latter study shows that only the homeodomain itself interacts directly with DNA. Our transgenic studies show that the Y54M alteration acted as a knockout, where ability to down-regulate ind and msh apparently is suppressed completely. The acidic domain deletion mutant showed limited down-regulation capability. By contrast, the tinman and the NK-2 box deletion mutants both behaved fully as functional vnd/NK-2 genes in their ability to repress ind and msh. Another single amino acid residue mutation in the homeodomain, H52R, stabilized the homeodomain both structurally and thermally. Nevertheless, this mutation in a transgenic experiment was toxic to the embryo and completely obviated growth as soon as expression of mRNA was initiated. The NMR determined tertiary structures of the Y54M vnd/NK-2 homeodomain in vitro both free and bound to DNA were compared with the wild type analog. The only structural difference observed for the mutant homeodomain was in the complex with DNA and involved a closer interaction of the methionine with A2 rather that C3 of the DNA. This change in the interaction of the homeodomain with DNA resulted in significant alteration in the lifetimes of the homeodomain-DNA complexes. The lifetimes were studied in four systems; 1) the wild type homeodomain bound to its cognate DNA, 2) the wild type homeodomain bound to a sequence of DNA that contains 5' - CAATGG - 3', which is the consensus sequence of homeodomains that contain methionine in position 54, 3) the Y54M homeodomain bound to wild type core DNA consensus sequence, and 4) the Y54M homeodomain bound to the DNA that contains 5' - CAATGG 3' as its core. The lifetimes of the wild type homeodomain bound to the wild type core DNA consensus sequence (i.e., #1) were at least one order of magnitude longer than possibilities 2 - 4. The implication or hypothesis here is that the lifetime of the transcriptional activation complex must be sufficiently long to insure proper embryonic development. To investigate this hypothesis further, CAT and beta-galactosidease reporter gene assays were carried out using several 750 base-pair sequences of the 5' upstream region of the vnd/NK-2 gene as promotors. Preliminary results demonstrated that alterations of either 5' - CAAGTG - 3' or 5' - CAATGG - 3' DNA fragments significantly altered the expression of both CAT and beta-galactosidease. Both activation and repression was seen depending on the specific changes made. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed upon ectopic expression of the Y54M vnd/NK-2 gene during embryogenesis. An important relationship between the regulatory role of the NK-2 class of homeobox genes in development and highly explicity homeodomain-DNA interactions is thereby established. These studies also demonstrate the relation between sequential and structural modularity with functional modularity, where in the above example, only the homeodomain appears to be necessary to down-regulate ind and msh. Studies on mutant CSX/NKX-2.5 homeodomains associated with atrial septum defect show that the tertiary structure of the Y54C mutant protein is not altered but that the interaction with the functional DNA binding sites is modified. The Y54C CSX/NKX-2.5 protein is totally non-functional. This modification of the interaction of the protein with the DNA likely constitutes the molecular basis of the disease. Recent structural studies suggest that tthat only part of the CSX/NKX-2.5 protein that is structured in the absence of binding to DNA or other protein factors is the homeodomain itself.