Student Theses and Dissertations
Structural Study of Disease Relevant ABC Transporters-Cystic Fibrosis Transmembrane Conductance Regulator and ABCA4
Date of Award
Doctor of Philosophy (PhD)
ATP-binding cassette (ABC) transporters are primary transporters that utilize the energy from ATP binding and hydrolysis to transport substrates across membrane against their concentration gradients . Structurally, canonical ABC transporters consist of four subunits— two transmembrane domains (TMDs) which form the substrate transport pathway and two nucleotide binding domains (NBDs) which dimerize upon ATP binding to provide energy for substrate transport. Most mammalian ABC transporters are exporters, with three exceptions: SUR—a regulatory protein for the KATP channel, cystic fibrosis transmembrane conductance regulator (CFTR)—an chloride channel and ABCA4 (aka the Rim protein and ABCR)—an retinylidene-PE importer [1-3]. In addition to their unique functional properties, both CFTR and ABCA4 are very important in human health. Mutations in CFTR cause cystic fibrosis, a lethal disease with a prevalence of 1 in 2,500 in Caucasian populations [4, 5]. Over 800 mutations have been identified in ABCA4 to associate with various types of retinal disease , including the Stargardt disease (also known as juvenile macular degeneration), the most common form of inherited macular degeneration [7, 8]. To understand how those two proteins function, I mainly took a structural approach to capture the structures of CFTR and ABCA4 in different functional states. In correlating the structures with functional data, we now have a deeper mechanistic understanding of these two important ABC transporters. The function of CFTR is regulated by ATP and phosphorylation. Once phosphorylated, ATP binding opens the CFTR channel and ATP hydrolysis closes it . First, we determined by cryo-electron microscopy (cryo-EM), the structure of dephosphorylated human CFTR in the absence of ATP (Chapter 2). With this structure, in conjunction with the functional studies performed by our collaborator (Prof. Laszlo Csanady and Prof. David C. Gadsby), we were able to propose a mechanism of how phosphorylation regulates CFTR. In addition, we identified a structural feature distinguishing CFTR from all other ABC transporters, which likely forms the structural basis for CFTR’s channel function. Next, we determined the structure of CFTR in the phosphorylated, ATP-bound state (Chapter 3 and 4). By comparing the ATP-free and -bound structures, we identified the nature of conformational changes that lead to channel opening. These structures also allow us to map many disease-causing mutants and explain how they lead to the malfunctioning of CFTR. To understand how small molecules, called potentiators, interact with CFTR and increase its open probability, we determined the structures of CFTR in complex with 2 potentiators— ivacaftor and GLPG1837 (Chapter 5). Interestingly, both small molecules bind to the same pocket inside the transmembrane region. These studies identified a hotspot on CFTR for rational drug design. Finally, I also studied ABCA4, the only known importer in mammalian ABC transporters. To understand how ABCA4 functions, I determined the structures of ABCA4 in the absence and presence of ATP (Chapter 6). Based on these structures, we propose a rudimentary transport mechanism for ABCA4. Future work will be carried out in the Chen lab to test this model.
Liu, Fangyu, "Structural Study of Disease Relevant ABC Transporters-Cystic Fibrosis Transmembrane Conductance Regulator and ABCA4" (2021). Student Theses and Dissertations. 645.
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy