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。此外，涉及三种保守区缺失突变体（即，tinman，编码NK-2特异性结构域的NK-2框，和编码酸性结构域的酸性框）和单个氨基酸残基置换。单个氨基酸残基置换涉及在同源结构域的第54位用甲硫氨酸置换酪氨酸（即，Y54M）。酪氨酸是负责识别含有5' - CAAGTG - 3'作为其核心的DNA序列的单个最重要的可变氨基酸残基。关于转基因研究，野生型和四个突变体vnd/NK-2基因的异位表达模式与两个下游靶基因ind和msh的表达一起进行了分析，这两个下游靶基因被vnd/NK-2下调。在检查的四个异位表达的突变基因中，只有Y 54 M改变充当敲除，其中下调ind和msh的能力显然被完全抑制。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的能力方面都表现为功能性vnd/NK-2基因。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-DNA复合物中的这种微妙变化导致Y 54 M vnd/NK-2同源结构域与DNA的结合亲和力的改变。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，CSX/NKX-2.5蛋白中只有一部分是在不与DNA或其他蛋白因子结合的情况下结构化的，这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. 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, the ectopic expression patters of the wild type and the four mutant vnd/NK-2 genes were analyzed together with the expression of two down-stream target genes, ind and msh, that are down-regulated by vnd/NK-2. Of the four ectopically expressed mutant genes examined, 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 subtle change in the homeodomain-DNA complex resulted in modifications of the binding affinities of the Y54M vnd/NK-2 homeodomain to DNA. 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.