3D STRUCTURE DETERMINATION OF RECOMBINANT BETA-1-GALACTOSYLTRANSFERASEFERASE

重组 β-1-半乳糖基转移酶的 3D 结构测定

• 批准号: 6100974
• 负责人: P K QASBA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100974
• 关键词: Escherichia coli; X ray crystallography; active sites; crystallization; enzyme activity; enzyme structure; galactosyltransferases; inclusion body; lactalbumin; method development; protein structure function; recombinant proteins; site directed mutagenesis; solubility; structural biology

The enzymes of galactosyltransferase family exhibit diverse functions, namely: they are involved in sugar transfer, cell-cell adhesion and pattern formation during development. The sugar acceptor and donor specificity of some of the enzymes can be modulated by alpha-lactalbumin (LA), a mammary gland specific calcium binding protein that has sequence and structural similarity to the c- type lysozymes (1). Since the enzymes of galactosyltransferase family are multifunctional, we are currently investigating the structure and function of various regions of the beta-1,4 galactosyltransferase(beta-1,4 GT) and its interactions with LA to account for the diverse functions. Currently, using the genetic engineering and crystal structure determination methods, the aim of our laboratory is to determine the three-dimensional structure of the catalytic domain, full length protein containing transmembrane (TM) domain and the complex beta-1,4 GT and its substrate modifier protein LA. The studies are aimed to define the sugar nucleotide and sugar acceptor binding specificities of the catalytic domain of beta-1,4 GT family, identify the region(s) that interact with LA in the presence of sugar which change the sugar acceptor specificity of the enzyme. Furthermore, our aim is to characterize the region that is involved in cell adhesion and interactions with laminin and other cell-matrix proteins. This structural information will be beneficial in understanding the role of glycosyltransferases at the cell surface. Earlier in our laboratory, the functional analysis of the TM domain was carried out by expressing and localizing the TM mutants in mammalian cells and some of the structural requirements of the TM domain were correlated with the function (2). For the structure and function analysis of the stem region, the sugar donor and acceptor binding regions of the catalytic domain, and for the studies on sugar-dependent protein-protein interactions between beta-1,4 GT and LA the recombinant proteins have been expressed in E.coli, proteins renatured from the inclusion bodies, folded in vitro, and analyzed for substrate bindings and enzymatic activities (3). These results have shown that the catalytic domain lies at the COOH-terminal portion of beta-1,4 GT and can be further subdivided into the N and C-terminal halves. The major binding region for the sugar acceptor lies in the N-terminal half of the catalytic domain, while the binding of the sugar nucleotide donor is localized to the C-terminal half, and in order that the catalysis to occur the two halves have to overlap at the catalytic surface. The O4-H of the GlcNAc has to be in the close proximity of the C1 of galactose (UDP-alpha-galactose) at the catalytic site, and the inversion of the configuration at C1 has to occur to generate a beta-glycosidic linkage. The disulfide bond between Cys 134 and Cys 247 is required during catalysis, but not for folding or binding substrates. The recombinant beta-1,4 GT and rat LAs, the short and long forms, and in vivo produced rat LAs have been crystalized for structure determination. The structure determination of some of these proteins are in progress.

半乳糖基转移酶家族的酶具有多种功能， 即：它们参与糖转移、细胞-细胞粘附和 在发展过程中形成的模式。糖受体和供体 某些酶的特异性可由α-乳白蛋白调节 (LA)，一种乳腺特异性钙结合蛋白，其具有序列 和C型溶菌酶的结构相似性（1）。以来 半乳糖基转移酶家族的酶是多功能的， 目前正在研究不同区域的结构和功能， β-1，4半乳糖基转移酶（β-1，4 GT）及其相互作用 与LA一起考虑不同的功能。目前，使用 基因工程和晶体结构测定方法，目的是 我们实验室的任务是确定 含跨膜蛋白的全长催化结构域 结构域和复合物β-1，4 GT及其底物修饰蛋白 路易斯安那州这些研究的目的是定义糖核苷酸和糖 β-1，4 GT催化结构域的受体结合特异性 家庭，确定与LA相互作用的区域， 改变酶的糖受体特异性的糖。 此外，我们的目标是描述所涉区域的特点， 细胞粘附和与层粘连蛋白和其他细胞基质的相互作用 proteins.这种结构信息将有利于 了解糖基转移酶在细胞表面的作用。 在我们实验室的早期，TM结构域的功能分析是 通过在哺乳动物中表达和定位TM突变体来进行 细胞和TM结构域的一些结构要求， 与函数（2）相关。对于结构和功能 茎区、糖供体和受体结合的分析 区域的催化结构域，并为糖依赖的研究 β-1，4 GT和LA之间的蛋白质-蛋白质相互作用 蛋白质已经在大肠杆菌中表达，蛋白质从 包涵体，体外折叠，并分析底物结合 酶活性（3）。这些结果表明， 催化结构域位于β-1，4 GT的COOH末端部分， 可以进一步细分为N 和C-末端的一半。主要 糖受体的结合区位于N-末端的一半， 催化结构域，而糖核苷酸供体的结合是 定位于C-末端的一半，为了使催化作用， 出现两个半部必须在催化表面重叠的情况。O4-H GlcNAc的C1必须非常接近半乳糖的C1， （UDP-α-半乳糖）在催化位点，和反转的 必须发生C1处的构型以产生β-糖苷键。 Cys 134和Cys 247之间的二硫键是在 催化，但不用于折叠或结合底物。重组 β-1，4-GT和大鼠LAs，短和长形式，以及体内产生的 大鼠LA已结晶用于结构测定。结构 其中一些蛋白质的测定正在进行中。