源自大肠杆菌的木糖：质子同转运蛋白的结构与功能研究

发布时间：2017-12-28 05:43

本文关键词：源自大肠杆菌的木糖：质子同转运蛋白的结构与功能研究　出处：《清华大学》2016年博士论文　论文类型：学位论文

更多相关文章： Xyl E MFS 膜转运蛋白 GLUT 转运机理

【摘要】：糖类代谢在新陈代谢等生命活动中具有关键作用,其中又以葡萄糖代谢最为重要。糖类进入细胞必须通过细胞膜上的糖转运蛋白。对葡萄糖来说,人体中有两类转运蛋白,与多种生理病理活动息息相关。本文选择其中一类GLUT家族蛋白作为研究对象,借助其原核同源蛋白Xyl E来考察。Xyl E源自大肠杆菌,是底物专一的木糖:质子同向转运蛋白,与I型GLUT家族成员有接近50%的一级序列相似度。葡萄糖可以抑制Xyl E的转运功能,不能被Xyl E转运;I型GLUT家族成员转运底物则不像Xyl E一样需要质子参与。因此Xyl E的转运机理仍然和I型GLUT家族成员存在一定差异。为了深入理解这类糖转运蛋白的机理,我们以X射线晶体学的手段,解析得到了Xyl E分别与底物木糖、底物类似物葡萄糖及人工合成的葡萄糖溴代衍生物结合的结构,分辨率分别为2.8?、2.9?和2.6?。这三个Xyl E结构呈现出经典的12次跨膜α螺旋MFS折叠方式,但在胞内区域多出一个四α螺旋的结构域,且具有向细胞外侧开放、部分封闭的全新构象,完善了该家族结构研究的未知信息。通过结构分析及生化实验验证,鉴别出了Xyl E在转运过程中识别底物的关键氨基酸残基。借助计算机软件同源建模的手段,建立人源GLUT1的三维蛋白结构模型。在该同源结构模型中,成功定位了一些I型GLUT家族成员疾病相关的突变残基,初步适用于致病机理研究。另外通过Xyl E和I型GLUT家族成员的序列比对分析,结合生化实验,进一步鉴定出Xyl E中第27位的天冬氨酸对于转运过程中质子耦联的调控作用。同样根据序列比对分析设计氨基酸突变,能改变Xyl E对底物的选择特异性,使其一定程度上具备转运葡萄糖的能力。以上研究开启了理解糖转运蛋白转运机制的新篇章,扩充了对MFS超家族蛋白的普遍认识,也为后续直接研究GLUT家族打下坚实基础。
[Abstract]:Carbohydrate metabolism plays a key role in metabolism and other life activities, and glucose metabolism is the most important. Sugar into cells must pass through the sugar transporter on the membrane of the cell. For glucose, there are two types of transporters in the human body, which are closely related to a variety of physiological and pathological activities. In this paper, one of the GLUT family proteins was selected as the research object and was examined with its prokaryotic homologous protein Xyl E. Xyl E, derived from Escherichia coli, is a xylose specific to the substrate: proton co transporter, which is similar to 50% of the I type GLUT family members. Glucose can inhibit the transport of Xyl E and can not be transported by Xyl E; the transport substrate of I type GLUT family, unlike Xyl E, requires proton participation. Therefore, the transport mechanism of Xyl E is still different from the members of the I type GLUT family. In order to further understand the mechanism of these sugar transporters, we have obtained the structure of Xyl E combined with substrate xylose, substrate analogues glucose and synthetic glucose bromine derivatives by X ray crystallography. The resolution is 2.8, 2.9 and 2.6, respectively. These three Xyl E structures show a classic 12 transmembrane alpha helical MFS folding mode, but in the intracellular region, there is a four alpha helix domain with a new conformation opening and partially closed to the cell, which improves the unknown information of the family structure. The key amino acid residues identified by Xyl E during the transport process were identified by structural analysis and biochemical test. With the help of computer software homologous modeling, a three-dimensional protein structure model of human GLUT1 was established. In this homologous structural model, a number of disease related mutations related to the I type GLUT family members have been successfully located, which are preliminarily applied to the study of pathogenic mechanism. In addition, through sequence alignment analysis of Xyl E and I GLUT family members, combined with biochemical experiments, we further identified the role of twenty-seventh aspartate in Xyl E in regulating proton coupling during transport. According to the sequence alignment analysis, the design of amino acid mutation can change the selectivity of Xyl E to the substrate, so that it has the ability to transport glucose to a certain extent. The above research opens a new chapter to understand the transport mechanism of sugar transporters, expands the universal understanding of MFS superfamily proteins, and lays a solid foundation for further study of GLUT family.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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