Three-dimensional Structures Of Biological Macromolecule

研究了NK-2类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，以及游离态和与DNA结合的全长CSX/NKX-2.5和NKX-3.1蛋白。在游离态和DNA结合态下，我们还研究了包含VND/NK-2同源结构域、连接子和NK-2特异性结构域的AD附加结构。后一项研究表明，只有同源结构域本身与DNA直接相互作用。我们的转基因研究表明，Y54M改变起到了敲除作用，下调ind和MSH的能力明显被完全抑制。酸性结构域缺失突变体的下调能力有限。相比之下，Tinman和NK-2盒缺失突变体在抑制IND和MSH的能力方面都完全表现为功能性的vND/NK-2基因。同源结构域的另一个单一氨基酸残基突变H52R在结构和热上都稳定了同源结构域。然而，转基因实验中的这种突变对胚胎是有毒的，一旦mRNA表达开始，就完全阻止了胚胎的生长。核磁共振测定的Y54M vnd/NK-2同源结构域在体外游离和与DNA结合的三级结构与野生型类似物进行了比较。唯一观察到的突变同源结构域的结构差异是与DNA的复合体，涉及蛋氨酸与DNA的A2而不是C3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了同源结构域-DNA复合体寿命的显著变化。在四个系统中研究了它们的寿命：1)野生型同源结构域与其同源DNA结合，2)野生型同源结构域与含有5‘-CAATGG-3’的DNA序列结合，5‘-CAATGG-3’是含有54位蛋氨酸的同源结构域的共同序列，3)Y54M同源结构域与野生型核心DNA共同序列结合，4)Y54M同源结构域结合以5‘-CAATGG 3’为核心的DNA。与野生型核心DNA一致序列(即#1)结合的野生型同源结构域的寿命至少比可能性2-4长一个数量级。这里的暗示或假设是，转录激活复合体的寿命必须足够长，以确保正常的胚胎发育。为了进一步研究这一假说，以vnd/NK-2基因5‘上游的750个碱基对序列作为启动子，进行了CAT和β-半乳糖苷酶报告基因分析。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。根据所做的具体改变，可以看到激活和抑制。这些由单个氨基酸残基替换引起的变化构成了Y54M vnd/NK-2基因在胚胎发育过程中异位表达时观察到的表型变化的分子基础。因此，在NK-2类同源框基因在发育中的调节作用和高度显性的同源结构域-DNA相互作用之间建立了重要的关系。这些研究还证明了序列模块化和结构模块化与功能模块化之间的关系，在上面的例子中，似乎只有同源结构域是下调ind和MSH所必需的。对与房间隔缺损相关的突变型CSX/NKX-2.5同源结构域的研究表明，Y54C突变蛋白的三级结构没有改变，但与功能DNA结合部位的相互作用发生了改变。Y54C CSX/NKX-2.5蛋白完全没有功能。这种蛋白质与DNA相互作用的修饰很可能构成了这种疾病的分子基础。最近的结构研究表明，在没有与DNA或其他蛋白质因子结合的情况下，CSX/NKX-2.5蛋白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.