...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508
【24h】

Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508

机译：P-糖蛋白的核苷酸结合结构域之间的通信通过涉及残基508的构象变化发生

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [H-3]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis. [References: 50]
机译：我们的目的是提供分子机制，以了解（i）两个核苷酸结合结构域（NBD）之间的相互作用以及（ii）NBD与P-糖蛋白（Pgp）内跨膜结构域之间的偶联的基本机制。为了促进这一点，我们在Pgp N末端NBD的表面暴露位置（E393C，S452C，I500C，N508C和K578C）引入了许多独特的半胱氨酸残基，这些残基先前经过工程设计可去除内源性半胱氨酸。使用基于原核NBD的晶体学特征的模型设计突变的位置。使用重组杆状病毒在昆虫细胞中表达单个半胱氨酸突变体，并借助多组氨酸标签通过金属亲和层析纯化蛋白质。根据药物刺激的ATP水解的特性判断，引入的半胱氨酸残基均未干扰Pgp的功能。通过与N-乙基-马来酰亚胺（NEM）共价结合的作用，研究了残基在Pgp催化循环中每个引入位点的作用。除一个突变（K578C）外，其他所有突变都可以用[H-3] -NEM标记。但是，仅在衍生的N508C亚型中观察到了ATPase活性的扰动。主要的功能表现形式是对ATP水解的“基本”速率的显着抑制。 NEM缀合的N508C亚型中，一系列药物可能会影响ATPase活性的程度和效力均未改变。结果表明，位于ATP水解过程中的构象变化会改变Pgp中NBD1的α-螺旋亚结构域中残基508的可及性。 [参考：50]

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号