Integrative structural and biochemical insights of COMATOSE and MsbA, two type IV ABC transporters

ATP-binding cassette (ABC) transporters are proteins that move a wide range of substrates across cellular membranes. Among them, type IV ABC exporters can play central roles in lipid handling, from the bacterial MsbA, which flips lipid A precursors across the inner membrane, to the plant peroxisomal transporter COMATOSE (CTS/AtABCD1), which delivers fatty acyl-CoAs for β-oxidation. Although decades of biochemical and genetic research have established CTS as indispensable for plant development, many important questions about its transport mechanism remain unanswered. By optimising expression and purification strategies, native CTS and catalytically inactive mutants were recovered in stable, active forms suitable for functional and structural studies. Activity assays confirmed ATPase activity in both detergent and lipid environments, providing a foundation for further exploration through transport assays. The reconstitution system allowed for the apparent binding affinity of substrate C18:0-ether-CoA to be reported. Integrative modelling using AlphaFold2/3 and docking simulations identified a conserved acyl-CoA binding pocket, where distinct residues appear to mediate substrate recognition and potentially contribute to the intrinsic thioesterase activity. Conservation analysis and comparisons with human homologues show evolutionary commonalities in substrate handling across the ABCD family. In parallel, this work evaluated emerging cryoEM technologies using the archetype transporter MsbA. It demonstrated the potential of new time-resolved cryoEM approaches to broaden the range of methods available for studying membrane proteins. These findings provide new mechanistic insights and set the stage for future high-resolution exploration of these challenging yet intriguing proteins.