Timing of CFTR Pore Opening and Structure of Its Transition State

Abstract

In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.

Exploring conformational equilibria of a heterodimeric ABC transporter

“…Based on our data we cannot rule out that the extracellular gate possibly covers a conformational ensemble between outward-occluded and outward-facing states. A recent biophysical study of CFTR elucidated a conformational progression of channel opening with the following sequence of events: NBD closure, propagation of the conformational change to the intracellular part of the TMDs and finally the opening of the extracellular gate (Sorum et al, 2015). The functional importance of extracellular gate opening in the context of substrate transport remains elusive and studying the effects of substrates or point mutations in the transporter will help to elucidate this intriguing aspect.…”

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

“…The combinations of mutations that were best for stabilizing NBD1, 6SS and 7SS, increased T m func from 22 °C for WT CFTR to 36 °C (Table 1). ATP binding and hydrolysis in CFTR, as in all ABC transporters, depends on the two NBDs interacting in a head-to-tail dimer to sandwich two MgATP molecules between the Walker A/B motifs of one NBD and the Signature motif of the other [32,66,67]. CFTR NBD1 has a degenerate Walker B motif and lacks the switch histidine (equivalent to H1402), making this composite site inactive [17,68].…”

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

“…Recent evidence from our group demonstrated that inhibition of CFTR affects HCO 3 -uptake resulting in low PKA activity and inhibition of cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction (9). It is well established in several systems that CFTR phosphorylation by PKA is required for its activation (15)(16)(17). For this reason, we postulated that an initial increase in PKA activity is necessary to activate CFTR channels and produce a sustained increase in HCO 3 -and cAMP.…”