Transmembrane helix 8 as the main allosteric mediator of CFTR gating revealed by molecular dynamics

Background The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel whose dysfunction underlies cystic fibrosis. Despite major advances in therapies, a detailed mechanistic understanding of CFTR gating remains essential for the rational development of next-generation drugs and for predicting mutation- and drug-specific effects. While recent cryo-EM structures captured ATP-bound, phosphorylated CFTR conformations, these experimental structures do not exhibit a continuous open chloride permeation pathway, limiting their direct use for gating analysis and in silico drug testing. Recently, open CFTR conformation was generated using molecular dynamics (MD) simulations at elevated membrane potential and characterized by a pronounced kink in transmembrane helix 8 (TM8) and a high channel open probability. Results We assessed the structural and dynamical validity of this open conformation using a combination of AlphaFold predictions, targeted MD, and extensive equilibrium MD simulations in lipid bilayers. Our results demonstrate that the TM8 is intrinsically highly dynamic and that the kink emerges spontaneously during equilibrium simulations. The open conformation has a substantially higher open probability as compared to that of the simulations initiated from the phosphorylated CFTR cryo-EM structure (6msm). Furthermore, in the open state, the catalytically active ATP-binding site 2 is allosterically coupled to TM8, based on analyses of residue motion correlations, suggesting a direct communication pathway between nucleotide binding and pore opening. Conclusions These results support the physiological relevance of the MD-generated open CFTR conformation and identify TM8 as a dynamic allosteric mediator linking nucleotide binding to pore opening, with lipid environment tuning the underlying gating mechanism.