EPS3.07 Mapping ivacaftor-induced structural changes in CFTR with computer simulations

Abstract

Ivacaftor, the first clinically approved cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, binds CFTR in a cleft formed by transmembrane segments 4, 5 and 8 at the protein-lipid interface. However, the conformational changes initiated by ivacaftor binding to CFTR remain largely unknown. Here, we investigated how the ivacaftor-binding site is allosterically coupled to functionally important regions of CFTR using a new computational approach named dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations. D-NEMD simulations combine molecular dynamics (MD) simulations under equilibrium and nonequilibrium conditions to map the evolving structural response of a protein to the removal of a ligand from its binding site. Using molecular models based on the cryo-EM structure of ivacaftor complexed with phosphorylated, ATP-bound human CFTR, which lack the R domain, we conducted extensive equilibrium MD simulations (five replicate 500 ns simulations, totalling 2.5 µs) and a large set of 410 short (5 ns) nonequilibrium simulations to identify conformational changes in CFTR elicited by the removal of ivacaftor from its binding site. The structural changes induced by ivacaftor removal started in its binding pocket formed by transmembrane segments 4, 5 and 8. Subsequently, they were gradually transmitted both upwards towards the extracellular vestibule of the CFTR pore and downwards through neighbouring transmembrane segments to reach the ATP-binding sites located at the dimer interface of the nucleotide-binding domains. Thus, our work reveals how ivacaftor-induced structural changes are propagated from its binding site to the ATP-binding sites and the CFTR pore. We will use this approach to understand how ivacaftor restores function to CFTR variants to inform the rational design of structure-guided therapies for cystic fibrosis.