Structural insights into the mechanism of a peroxisomal ABC transporter

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 objectives
1. Purify wild type and two mutant forms of AtABCD1 which are predicted to correspond to apo and ATP bound pre hydrolysis states
2. Determine the structure by cryoEM in the presence of different substrates/inhibitors
3. 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.