The CFTR plays a crucial regulatory role in ion/fluid homeostasis essential to lung health, impacting both anion (directly) and cation (indirectly) transport. The complexity of CFTR function requires a complex and dynamic structure that has evolved relatively recently as a unique ABC transporter family member. Like all proteins, CFTR evolution had to achieve a balance between structural complexity and thermodynamic stability with the result that only ~25% of wild-type polypeptide chains synthesized achieve a stable state. The AF508 mutation exacerbates inefficient biogenesis, reducing this proportion to essentially zero. Some manipulations including reduced temperature, down-regulation of quality control components and so-called "corrector" small molecules improve cellular processing and trafficking. However, these maneuvers do not restore Thermal Stability (TS) and the partial channel function at temperatures <30oC is rapidly lost at 37oC. Thus effective therapies require reagents that restore TS. Here we focus on the allosteric coupling pathways that determine TS to understand its mechanistic basis so that it can be manipulated rationally. SA 1 will elucidate the destabilizing influence of the Regulatory Insertion (Rl) in NBD1 and how its excision restores stability and function. SA 2 will employ two complementary approaches to identify other changes that restoreTS, the first utilizing molecular dynamics and the second exploring the basis of retained TS by AF508 CFTRs of some other species that are stable. These methods have already revealed that the introduction of proline residues at key positions in coupling pathways stabilize AF508 CFTR. SA 3 will utilize both small and larger molecule binders of CFTR as thermal stabilizers. First, small molecule libraries will be screened computationally (validated experimentally) for binding to regions of the protein implicated in TS. Second, we will identify larger protein/peptide binders including synthetic antibodies and nanobodies. Effective initial reagents have already been developed in collaboration with leading laboratories in both fields. In addition to providing an entirely new paradigm with integrated computational and experimental approaches to the CF problem, we provide the other projects of this PPG with essential CFTR tools and methodologies.
The proposed studies are specifically relevant to cystic fibrosis as they focus on a here-to-for neglected fundamental aspect of the molecular defect. As CFTR is also sensitive to environmental insults of the lung that contribute to COPD, the project is also relevant to that major health problem. Even more broadly, CFTR is also important in the functioning of the intestinal and reproductive tracts and other tissues such as the sweat gland, important in body temperature control.
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