Structural insights into the mechanism of a peroxisomal ABC transporter

ABC转运体将ATP水解偶联到各种底物跨膜的运动中，并且存在于所有生物和所有膜中。过氧化物酶体ABC转运体将β氧化途径的底物从细胞质转运到过氧化物酶体。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，一个单一的广谱转运蛋白接受多种酰基辅酶a底物并在运输过程中对其进行切割（这种ABC转运蛋白的独特活性）。在人类中，有三种不同的转运蛋白，它们具有更明确但重叠的特异性。重要的问题仍然没有答案，是什么决定了底物特异性，为什么它在不同的生物之间不同，酰基辅酶a硫酯酶活性的结构基础是什么，以及它是如何在运输循环中整合的？该小组之前的工作已经建立了拟南芥过氧化物酶体ABC转运体的稳健表达、纯化方案和功能。因此，这个项目将围绕以下目标展开：纯化野生型和两种突变型AtABCD1，预测它们对应于载脂蛋白和ATP结合的预水解状态2。在不同底物/抑制剂存在的情况下，用低温电镜测定其结构。将结构与生化分析确定的转运周期联系起来。这些目标的完成将为我们的理解提供一个转变，不仅在这些重要的转运体的结构方面，而且揭示了底物运输和切割的机制。

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.