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

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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 Ã… and a recently-obtained higher-resolution structure (at 6.5 Ã…) 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.

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1. COBALT-cftr-orthologs.pdf : Result of a Clustal alignment of 39 CFTR (ABCC7) orthologs including the human ortholog. The main text in the thesis refers to residues that are highly conserved across the orthologs but are unique to CFTR within the ABCC family (see 2 below) and which may therefore be crucial for the evolution of CFTR channel function. 2. Cobalt-abcc.pdf: Result of a Clustal alignment of the human CFTR (ABCC7) sequence with 19 different ABC transporter family members showing high sequence conservation to CFTR. Of the 19 closest hits in terms of sequence homology, all are ABCC family members except YCF1, a yeast cadmium resistance factor. The main text refers to residues that are unique to CFTR within this clustal alignment (with the hypothesis that these residues may be linked to the evolution of CFTR as a channel).3. Cmlsrev-ford-b.docx: Review recently accepted for publication in Cellular and Molecular Life Sciences with referees’ comments taken into account. 4. escreen_TOP96_20150417093414_A1 & A7: Details of the screen conditions based on the Well A1 and A7 in the TOP96 screen in the 3D crystallization trials

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