Cystic fibrosis (CF) is the most common human genetic disease, occurring prevalently in the Caucasian population at a rate of 1 to 2500 newborns. It is an autosomal recessive disease caused by mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, which encodes a chloride channel expressed mainly in epithelial cells, but which is also involved in the bicarbonate–chloride exchange. The most common CF symptoms include progressive lung disease and chronic problems of the digestive apparatus (Riordan et al., 1989), whose degree of severity depends on other genetic and/or environmental factors. CF pathogenesis is characterised by the build-up of thick, sticky mucus in multiple mucin-producing organs, such as lungs, sinuses, intestine, pancreas and reproductive organs. For this reason, CF is also denominated mucoviscidosis, implying that mucins - polymeric, gel-forming O-linked glycoproteins responsible for the viscoelastic properties of the mucus - play a critical role in the disease (Kreda et al., 2012).
The aim of the present Ph. D. work was to investigate the structural features of two CFTR domains: the nucleotide binding domains (NBDs) - responsible for the gating mechanisms of the channel, and which have been proposed to serve as drug targets - and the regulatory domain (RD), directly involved in the activation of the channel. Knowledge of these aspects could likely improve understanding of the aberrant functionality of defective CFTR, and also help designing therapeutic strategies to either correct the defective protein in situ, and/or to potentiate its physiologic channel activity.
The present thesis refers essentially to the four published papers containing most of the results obtained during the 3 year-doctorate course. The first one reports on some biochemical and structural features of NBDs, which were investigated using biochemical assays and measures of small angle x ray scattering (SAXS), while the second paper dealt with the interaction of NBDs with a potentiator (2-pyrimidin-7,8-benzoflavone, PBF) of CFTR activity. Instead, the third and the fourth papers considered RD under non-phosphorylated and phosphorylated conditions, and the influence of phosphorylation on the conformation of the domain as followed by circular dichroism (CD) and SAXS experiments.
Briefly, the obtained results allowed us to draw the following principal conclusions.
NBDs When in an equimolar mixture and in the presence of ATP, NBDs form a dimer, whose conformation can be significantly changed by PBF. In addition, data could be exploited to reconstruct the ab-initio model of NBDs both as dimer (with or without PBF) and as isolated monomers.
RD In this case, obtained results on biochemical, structural and thermodynamic RD aspects allowed us to reconstructing a low-resolution, 3-D model of the native and phosphorylated protein, and to underline how phosphorylation induces the conformational change of the domain and the decreasing of RD stability
