(685) CryoEM reconstruction of the conformational ensemble adopted by hCFTR-G551D provides insight into the folding pathway and the mechanism-of-action of the dual corrector/potentiator Elexacaftor (VX445)
Professor Columbia Iniversity New York, New York, United States
Background: Cystic fibrosis (CF), the most common fatal human genetic disease in Caucasian populations, is caused by a wide spectrum of mutations in an ATP-gated chloride ion channel called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Empirical screening for compounds enhancing CFTR activity in tissue-culture cells led to the development of a triple-drug combination therapy that effectively cures CF in the vast majority of patients. The lynchpin of this therapy is Elexacaftor (VX445), a profoundly hydrophobic compound that corrects the folding defect caused by some prevalent CFTR mutations and also potentiates the activation of chloride channel conductance in properly folded CFTR.
Methods: We have employed Global Conformational Ensemble Reconstruction (GCER) to analyze the distribution of conformations adopted by human CFTR molecules harboring the G551D disease-causing mutation following purification in digitonin/cholesterol mixtures.
Results: These studies identify a high-affinity binding site for Elexacaftor in a region of the protein that is allosterically coupled to both the interface between the protein's first nucleotide-binding domain (NBD1) its transmembrane domain and also the exit pore of the chloride channel in its transmembrane domain. They furthermore reveal that purified CFTR molecules dynamically sample a diversity of different conformational states, including a stable state reconstructed at 3.3 Å resolution in which one-third of its first transmembrane domain is partially unfolded.
Conclusions: The structural characteristics of this state, combined with the conformational properties of the other states in the observed conformational ensembles, provide a detailed structural and thermodynamic model for the mechanism by which Elexacaftor corrects folding defects and and activates the conductance of CFTR channels.
Acknowledgements: Sergey M. Vorobiev1¶, Chi Wang1¶, ZhengrongYang2¶, Jiang Fan2, John N. Wehby2, Andrei A. Aleksandrov3, Blaine Loughlin1, Oliver B. Clarke1,Robert A. Grassucci1,Kam-Ho Wong1, QingxianZhou2; ShikhaSingh1, Jack Riordan3, Ina L. Urbatsch4, Christie Brouillette2, John C. Kappes2, Joachim Frank1, & John F. Hunt
