Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460–1348 and 460–1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460–1348 or positions 460–1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…This model envisages "limited motions" of the NBD dimer in the completion of one catalysis (and gating) cycle. Of note, this model is supported by a report that employed a completely independent approach (Szollosi et al 2011). The significance of this new model lies in the fact that all members of the ABCC subfamily possess only one catalysis-competent site in their NBDs.…”

Long‐range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels

“…The P o for each subsequent condition was then calculated by dividing the NP o product measured for that condition by this value. Second, stationary noise analysis was used to estimate P o s from multichannel records as previously described for CFTR (30, 31). This method is based on the premise that the currents that are mediated by channels with high P o s exhibit less variance (σ 2 ) than those mediated by channels with lower P o s, which gate more dynamically.…”

“…This method is based on the premise that the currents that are mediated by channels with high P o s exhibit less variance (σ 2 ) than those mediated by channels with lower P o s, which gate more dynamically. For each current record, the mean current (m) and the current variance (σ 2 ) were obtained from Clampfit after the baseline noise was subtracted (30, 31). The P o for each patch was estimated from these parameters using the equation μ (1 – P o ) = σ 2 /i , where i is the unitary current at the experimental holding potential (–0.4 pA at –60 mV for WT‐CFTR).…”

Nonequilibrium Gating of CFTR on an Equilibrium Theme

“…This model was also supported by a report employing an independent approach. By studying the energetic coupling between different residue pairs located around ABP1, Szollosi et al (69) found that the coupling energy between these residue pairs does not change throughout the gating cycle. Since each pair tested includes one residue in the head of NBD1 and the other in the tail of NBD2, these results indicate that interaction of two NBDs around ABP1 remains static during gating.…”

“…Since each pair tested includes one residue in the head of NBD1 and the other in the tail of NBD2, these results indicate that interaction of two NBDs around ABP1 remains static during gating. (69). Of note, like CFTR, many members in the ABC protein family contain only one catalysis-competent site (32, 39, 52, 72).…”