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CSX/NKX-2.5和NKX-3.1蛋白的全长，无论是在自由状态还是与DNA结合状态。此外，还进行了涉及3个保守区缺失突变体（tinman、编码NK-2特异性结构域的NK-2 box、编码酸性结构域的酸性box）和单个氨基酸残基置换的转基因研究。单氨基酸残基替换包括用同位结构域（即Y54M）第54位的蛋氨酸替换酪氨酸。酪氨酸是唯一最重要的可变氨基酸残基，负责识别包含5‘ - CAAGTG - 3’为核心的DNA序列。在转基因研究中，只有Y54M的改变起到了敲除作用，其下调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的能力上都完全表现为功能性vnd/NK-2基因。核磁共振测定了体外游离和结合的Y54M vnd/NK-2同源结构域的三级结构，并与野生型类似物进行了比较。突变体同源结构域唯一的结构差异是在与DNA的复合体中，并且涉及到蛋氨酸与A2而不是DNA的C3的更密切的相互作用。同位结构域与DNA相互作用的这种变化导致同位结构域-DNA复合物寿命的显著改变。研究了四种体系的寿命；1)野生型同源结构域与同源DNA结合，2)野生型同源结构域与含有5‘ - CAATGG - 3’的DNA序列结合，这是54号位置含有蛋氨酸的同源结构域的一致序列，3)Y54M同源结构域与野生型核心DNA一致序列结合，4)Y54M同源结构域与含有5‘ - CAATGG 3’为核心的DNA结合。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'上游区域的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检测。初步结果表明，5‘ - CAAGTG - 3’或5' - caaggg - 3' DNA片段的改变显著改变了CAT和β -半乳糖苷酶的表达。激活和抑制取决于所做的具体改变。这些由单个氨基酸残基替换引起的变化构成了胚胎发生期间Y54M vnd/NK-2基因异位表达所观察到的表型改变的分子基础。NK-2类同源盒基因在发育中的调控作用与高度显式同源结构域- dna相互作用之间的重要关系由此建立。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，只有同域似乎是下调ind和msh所必需的。对与房间隔缺损相关的突变体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，但与功能性DNA结合位点的相互作用发生了改变。Y54C CSX/NKX-2.5蛋白完全无功能。这种蛋白质与DNA相互作用的改变可能构成了这种疾病的分子基础。最近的结构研究表明，CSX/NKX-2.5蛋白中只有一部分是在不与DNA或其他蛋白质因子结合的情况下结构的，即同源结构域本身。

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. In addition, transgenic studies involving three conserved region deletion mutants (i.e., tinman, the NK-2 box that encodes the NK-2 specific domain, and the acidic box that encodes the acidic domain) and a single amino acid residue replacement were carried out. The single amino acid residue replacement involved replacing tyrosine with methionine in position 54 of the homeodomain (i.e., Y54M). The tyrosine is the single most important variable amino acid residue responsible for the recognition of a DNA sequence that contains 5' - CAAGTG - 3' as its core. With regard to the transgenic studies, only 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. 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 that 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.