Integrating the Signals: Implications of CFTR NBD1 Allostery to Cystic Fibrosis

Abstract

Cystic fibrosis-causing mutations in CFTR can lead to protein misprocessing or channel gating defects, perturbing the ion and fluid flow across epithelial surfaces. CFTR transmembrane helices extend into the cytoplasm and interface with two nucleotide-binding domains, NBD1 and NBD2, via short 'coupling helices' (CLs). Channel gating is regulated by ATP binding and hydrolysis in the NBD1/NBD2 dimer and by phosphorylation of the NBD1 regulatory insert (RI) and R region immediately C-terminal of NBD1. We have used NMR spectroscopy to probe changes in conformational populations caused by ligand binding or mutation, revealing an allosteric network linking the CL4 binding site to regions near the RI and the R region. Conformational exchange occurs on multiple timescales in the NBD1 αRI construct, including μs-ms exchange and elevated flexibility in loops and C-terminal helices H8 and H9. Q637R, a mutation between H8 and H9 near to where the R region begins, changes dynamics in the RI deletion site and the CL4 binding site. VX-809, a drug that partially rescues both folding and gating defects, binds to the β-strands beneath H8/H9 of full-length NBD1, destabilizing these helices and perturbing some chemical shifts in the CL4 binding site. Titration of a CL4 peptide results in remote changes to H8/H9 and correlated chemical shifts observed at the RI. An allosteric network coupling the CL4 binding site to regions near both of the phospho-regulatory sites may contribute to the folding and gating defects that arise from mutations at the CL4:NBD1 interface, including those of the most common CF-causing mutation, F508del. Coupling of remote NBD1 sites by the observed allosteric network suggests that distinct inputs are integrated in CFTR processing and function and implies that correlation of NBD-ICL interactions might be achieved through targeting of these allosterically-linked sites.