Linking the Catalytic Cycle of the Nucleotide Binding Domains to Channel Gating in CFTR

Abstract

The chloride channel CFTR is an ATP Binding Cassette (ABC) protein, a member of a family of active transport proteins. Following a mechanism conserved among all ABC proteins, binding of ATP to CFTR's two cytosolic nucleotide binding domains (NBDs) induces formation of a stable intramolecular NBD1/NBD2 dimer with two ATPs occluded at the interface, and ATP hydrolysis disrupts this dimer. While in homologous active transporters dynamic formation/dissociation of the NBD dimer is coupled to flipping between inward- and outward-facing TMD conformations, in CFTR these events are coupled to opening/closure of the chloride permeation pathway, allowing real-time detection of these conformational events: using single-channel patch-clamp recordings we seek to understand the precise timing and direction of molecular motions associated with each gating step. A limitation for reconstructing timing is that conformational transitions that are not associated with pore opening/closure go undetected in our recordings. However, information on these steps is hidden in the distributions of open (burst) and closed (interburst) dwell times. Our studies on the distributions of burst durations identified two kinetically distinct open states (pre- and posthydrolytic), and provided estimates for their life times. The peaked shape of the distribution violates microscopic reversibility and suggests nonequilibrium gating for wild-type CFTR, with pore closure strictly coupled to ATP hydrolysis. This coupling is partially or fully disrupted in various catalytic site mutants. Burst distributions obtained in the presence of ATP analogs, or for NBD1 mutants, suggest a crosstalk between the two ATP binding sites. The direction of motions can be studied by detecting gating-associated changes in energetic coupling between select position pairs. Using thermodynamic mutant cycles we have identified a conserved pair of residues within NBD2 which appears to form a molecular switch that signals ATP binding.