Functional and structural studies of the human ABC transporter ABCB8

• Main Author: Faust, Belinda Raffaella
• Other Authors: Carpenter, Elisabeth ; Shintre, Chitra
• Published: University of Oxford 2017
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.757776

ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins and are ATP-driven transport systems, which are found universally in all pro-and eukaryotes. Human ABC transporters have key roles in many physiological processes in the plasma membrane and within membranes of cell organelles, such as peptide transport, lipid flipping and multi-drug resistance. ABCB8, a sub-family B ABC transporter of the inner mitochondrial membrane, was found to be crucial for normal cardiac function, regulation of mitochondrial iron levels and cytosolic iron-sulphur cluster enzyme function and oxidative stress. Impaired function of ABCB8 resulted in mitochondrial iron accumulation, oxidative stress and cell death. In this thesis I developed a protocol for the purification of human ABCB8 providing yields and stable protein sufficient for structural studies using X-ray crystallography. Strategic optimisation of the purification protocol and crystallisation methods, resulted in crystals that diffracted to a resolution of 3.8 Å and allowed me to solve the first structure of human ABCB8. The ABCB8 structure adopted an outward-facing conformation that resembles a post-ATP-hydrolysis state in the alternating access mechanism, that was not yet observed in type I ABC exporters. In addition to the structural studies on ABCB8, I set out to explore the effects of ATP analogues, ATP transition state analogues and ADP on purified ABCB8 with biochemical and biophysical methods. Furthermore, I find that ATPase activity of ABCB8 in proteoliposomes is dependent on cardiolipin (CDL). The first structure of ABCB8 in an outward-facing conformation gives new insights into the alternating-access mechanism of subfamily B ABC transporters. Moreover, the purification and reconstitution protocol provided in this thesis is the foundation for further studies aiming to understand ABCB8's function in more detail.