Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

Abstract

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

“…For example, the generally accepted mechanistic model of CFTR envisages the degenerate site to be constantly closed during the entire gating cycle, a notion supported by the fact that nucleotides bind more tightly to the degenerate than to the consensus site of CFTR (28). Despite some disputes whether the degenerate site changes its conformation as the channel progresses through its states (29,30), there is unanimous agreement that inter-NBD contacts need to be established at all times to explain the experimentally observed cross-talk between the degenerate and the consensus site. As suggested recently (30), a partially opened degenerate site as seen in TM287/288 does not contradict current functional models of CFTR, but rather could explain how the ATP binding sites sustain their ability to cross-communicate while the TMDs adopt an inward-facing closed-channel state.…”

Stoichiometry and novel gating mechanism within the cystic fibrosis transmembrane conductance regulator channel

“…During the gating, one nucleotide‐binding site of NBDs remains associated for several gating cycles by binding ATP, while the other catalytically active binding site cycles between a closed state binding ATP (C 1 ), a non‐hydrolytic open state (O 1 ), a posthydrolytic open state (O 2 ) and a closed state binding ADP (C 2 ; Csanády et al . , ). In our hypothesized synergistic gating model, one CFTR channel (T338A/T339A in Fig.…”

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

“…A well studied example of a eukaryotic protein possessing a degenerate NBS is the cystic fibrosis transmembrane regulator. Here, site 1 (degenerate) is proposed to be bound to ATP through many cycles of ATP hydrolysis occurring at site 2 (canonical) and thus play a supporting role (16). This leads us to question the functional relevance for the protein and on a broader scale to the cell for maintaining a degenerate NBS, because such evolutionary conservation of this trait in yeast ABC transporters does need an explanation.…”