Structural insights into the mechanism of a peroxisomal ABC transporter

过氧化物酶体 ABC 转运蛋白机制的结构见解

• 批准号: 2601483
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2601483
• 关键词: Structural; insights; into; mechanism; peroxisomal

ABC transporters couple ATP hydrolysis to the movement of diverse substrates across membranes and are found in all organisms and all membranes. Peroxisomal ABC transporters transport substrates for the beta oxidation pathway from the cytosol into the peroxisome. Genetic studies show they are of profound importance for growth and development of the organism. In plants a single broad specificity transporter accepts a diverse range of acylCoA substrates and cleaves them upon transport (a unique activity of this ABC transporter). In humans there are 3 distinct transporters with more defined but overlapping specificity. Important questions that remain unanswered are what determines the substrate specificity, and why it differs between organisms, what is the structural basis of the acyl coA thioesterase activity and how is it integrated in the transport cycle? Previous work from the group has established robust expression, purification protocols and functional for the peroxisomal ABC transporter from Arabidopsis thaliana. Therefore, this project will be based around the following objectives1. Purify wild type and two mutant forms of AtABCD1 which are predicted to correspond to apo and ATP bound pre hydrolysis states2. Determine the structure by cryoEM in the presence of different substrates/inhibitors3. Relate structures to the transport cycle as determined by biochemical assays.Completion of these objectives will provide a shift in our understanding not just on the structural aspects of these important transporters but also reveal the mechanism for substrate transport and cleavage.

ABC转运蛋白将ATP水解酶与不同底物跨膜运动相结合，在所有生物体和所有膜中都存在。过氧酶体ABC转运蛋白将β氧化途径的底物从胞浆输送到过氧酶体。遗传学研究表明，它们对生物体的生长和发育具有深远的重要性。在植物中，单一的广泛的特异性转运体接受不同范围的酰基辅酶A底物，并在运输时将它们切割(这种ABC转运体的独特活动)。在人类中，有三种不同的转运蛋白，具有更明确但重叠的特异性。尚未回答的重要问题是：是什么决定了底物的专一性，为什么不同生物体的底物专一性不同，酰基辅酶A硫代酯酶活性的结构基础是什么，它是如何整合到运输周期中的？该小组之前的工作已经为拟南芥的过氧体ABC转运蛋白建立了强大的表达、纯化方案和功能。因此，该项目将围绕以下目标1展开。纯化野生型和两种突变形式的AtABCD1，它们被预测对应于apo和ATP结合的预水解状态2。在不同底物/抑制剂存在的情况下，通过冷冻EM确定结构3。将结构与生化分析所确定的运输周期联系起来。完成这些目标不仅将使我们对这些重要转运蛋白的结构方面的理解发生转变，而且还将揭示底物运输和切割的机制。