Thermal stability and structural studies of the human Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) is a member of the ATP-binding cassette (ABC) membrane transporter family that has evolved as an ion channel which is directly linked to the disease cystic fibrosis (CF). Cystic fibrosis, the most common inherited disease in populations of European descent, is due to mutations causing loss of CFTR function. Drugs targeting some channel-disrupting mutations such as G551D have recently been approved, but a structure for CFTR that could aid drug development is needed. In this thesis, G551D, the second most frequent CF missense mutation was chosen for study because of its affect on gating. The main aims were to compare the mutated protein with wild-type CFTR and other mutations; to use the protein as a negative control in assays of CFTR function and lastly to lock the CFTR protein in a closed state. The latter aim was pursued in order to test the hypothesis that a locked channel could be more stable and hence better suited for biophysical and structural studies including the crystallization of the protein. This thesis presents the generation, expression and purification of milligram quantities of the human G551D CFTR protein in a Saccharomyces cerevisiae (yeast) expression system. Two detergents, dodecylmaltoside (DDM) and lyso-phosphatidyl glycerol (LPG), were used to solubilize and extract CFTR and subsequent purification was by nickel affinity, FLAG affinity and size exclusion chromatography (SEC). In LPG, approximately 20 mg CFTR was recovered from an 18 L fermenter. Thermal stability of wild-type CFTR and the effects of different human CFTR mutations were studied using two different measurements, thermal gel analysis and coumarin maleimide binding (CPM binding). In the thermal stability studies, the additional effects of the two approved CFTR drugs, VX-770 and VX-809, were studied. G551D CFTR was shown to be more stable than WT and F508del CFTR. CFTR purified in the two detergents was also compared using the thermal stability assay and the relationship between stability and structure/function will be discussed. The CPM assay presented here was developed into a medium-throughput assay of potential use for CFTR modulator drug screening. The LPG-purified CFTR was a homogenous population of monomeric particles as judged by electron microscopy (EM) studies and could be concentrated to up to 30 mg/ml. A low resolution structure of G551D CFTR was initially generated by single-particle cryo-EM at about 13 A and a recently-obtained higher-resolution structure (at 6.5 A) will also be presented and discussed. The structures show that G551D CFTR displays an outward-facing conformation even in the absence of nucleotide (ATP), which is a novel finding for ABC transporters. The two nucleotide-binding domains interact closely over a wide interface. LPG-purified G551D CFTR was also used in the 3D crystallization trials and the prospects for using an X-ray crystallographic approach for structure determination are discussed.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Ford (Supervisor) & Stephen Prince (Supervisor)