Dynamical Effects of Mutations and Ivacaftor Binding on the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

Cystic fibrosis (CF) is a life-shortening autosomal-recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel responsible for chloride and bicarbonate transport. Dysfunctional CFTR leads to mucus accumulation, chronic infection and progressive organ damage. Although small-molecule modulators, such as potentiators (ivacaftor) and correctors (lumacaftor, tezacaftor), have improved clinical outcomes, they often only partially restore CFTR function, and the precise atomistic mechanisms underlying their effects remain unclear.
In this thesis, I employed extensive molecular dynamics (MD) simulations to investigate the structural and dynamic effects of four clinically relevant CFTR mutations (G551D, G1349D, S549N and F508del) and to characterise ivacaftor’s molecular actions. I developed a comprehensive simulation pipeline integrating high-resolution cryo-EM structures with AlphaFold2 predictions, producing 32.5 μs of MD trajectories for both wild-type (WT) and mutant CFTR variants. Simulations revealed mutation-specific disruptions of local dynamics and ATP-binding geometry, both of which in G551D and G1349D were partly rescued by aspartate protonation. Ivacaftor predominantly influences the membrane-spanning domain, producing only subtle changes in ATP-binding interactions without significantly altering overall nucleotide-binding domains separation, consistent with an allosteric mode of action.
Simulations of both WT and mutant CFTR showed stable chloride occupancy within the pore and continuous bidirectional water permeation, highlighting residues likely essential for gating and ion conduction. However, full chloride passage likely requires longer-timescale structural transitions not captured here.
Complementary dynamical-nonequilibrium MD (D-NEMD) simulations captured rapid conformational responses following ivacaftor removal, identifying mutation-specific rearrangements involving transmembrane helices M5 and M8, critical for allosteric communication. This analysis produced mutation-dependent allosteric “fingerprints”, providing mechanistic insights into differential responses to drug binding.
Collectively, these findings deepen our molecular understanding of CFTR dysfunction and modulation and will likely contribute to structure-based drug development for CF.

Date of Award	30 Sept 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Marc W Van der Kamp (Supervisor) & Deborah K Shoemark (Supervisor)