Molecular dynamics study of Cl− permeation through cystic fibrosis transmembrane conductance regulator (CFTR)

Abstract

The recent elucidation of atomistic structures of Cl− channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl− ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl− translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl− translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl− ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl− pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

Computational analysis of long-range allosteric communications in CFTR

“…We observed very similar conformational homogeneity and stability with another member of the ABC transporter family (58). Along the same lines, MD simulations showed that the closed state with dimerized NBDs is stable over hundreds of nanoseconds (79).…”

“…We observed very similar conformational homogeneity and stability with another member of the ABC transporter family (58). Along the same lines, MD simulations showed that the closed state with dimerized NBDs is stable over hundreds of nanoseconds (79). Taken together, these results strongly argue in favor of an induced-fit mechanism, and against MWC/ensemble-selection/population shift models which assume pre-existence of multiple conformations and a spontaneous dynamic equilibrium between them).…”

“…Several lines of evidence suggest that the final missing component that enables conductance are the dynamic fluctuations of the NBDs-closed state: MD simulations of chloride conductance showed that a major conformational change is not required for channel opening. Instead, slight side chain re-orientations of pore residues enable sporadic pulses of chloride conductance (79). Single molecule measurements also failed to identify any conformational changes between the conducting and non-conducting states (70).…”

Probing Phosphorylation-Induced Vibrational Couplings in CFTR by 2D IR Spectra Simulations

“…Phosphorylation was achieved by replacing the hydroxyl hydrogen on the serine side chain with phosphite (-PO 3 2- ). The DOPC lipid bilayer (44) was used to simulate the cytoplasmic membrane environment, and in our work, it has been expanded to a system with 1152 lipids and 43425 water molecules. A 20 ns simulation at 310 K has been used to stabilize this expanded system.…”

Structural identification of a selectivity filter in CFTR

“…Whereas the intracellular entrance (38, 39) and the cytosolic vestibule (42–46) of the pore were clearly defined in the published structures of CFTR (40, 47–49), the exact path of chloride from the inner vestibule to the extracellular space remains unclear. Extensive mapping by mutagenesis (50–53), cysteine accessibility (38, 54–56), and molecular dynamics (57) has also not allowed unambiguous assignment of the extracellular exit. Furthermore, although many residues have been shown to influence the conductance or relative permeabilities of different anions in CFTR (reviewed in (19, 20, 41)), a specific selectivity filter has yet to be defined.…”