Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

“…Thus, one can envision that, if the opening of G551D-CFTR also involves NBD dimerization, a more hydrophobic ligand in the binding site(s) may facilitate dimerization of the two NBDs. In support of this notion, the maximal opening rate of WT-CFTR with P-ATP is slightly higher than that of ATP (44,45).…”

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.…”

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

“…It is thus not surprising that site 1 was shown to have a very low nucleotide turnover rate (15,(27)(28)(29). However, except for this low ATPase activity, it is unclear whether some other properties of a canonical composite site have been affected by the aforementioned non-consensus substitutions or uncommon residues in CFTR site 1.…”