Extent of Nucleotide-Binding Domain (NBD) Separation When a CFTR Channel Closes

Artur, Luiz; Chaves, Poletto; Gadsby, David C.

doi:10.1016/j.bpj.2010.12.1662

Cited by 4 publications

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“…What mechanistic insight can be gained from these observations? The recent suggestion that composite site 1 of CFTR does not separate upon channel closure (Tsai et al, 2010; Szollosi et al, 2011) has fostered a view of the degenerate site as a simple rigid scaffold, which does not participate in the functional movements of asymmetric ABC-C proteins (although this proposal has remained a matter of contention; Artur et al, 2011). Our present data are hard to reconcile with such an assignment.…”

Section: Discussionmentioning

confidence: 99%

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

Csanády

Mihályi

Szöllősi

et al. 2013

Journal of General Physiology

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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.

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Section: Discussionmentioning

confidence: 99%

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

Csanády

Mihályi

Szöllősi

et al. 2013

Journal of General Physiology

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“…These findings provide independent support for the model recently proposed by Tsai et al (2009, 2010), who hypothesized that the NBD dimer only partially separates after ATP hydrolysis. Preliminary ATP binding at NBD1 triggers formation of a tight dimer interface around the inactive composite site 1, which is then maintained during several channel gating cycles (but compare Poletto Chaves and Gadsby, 2011). This proposal is consistent with, and refines, earlier models (Basso et al, 2003; Vergani et al, 2003) in which the conformational changes at the NBDs associated with channel gating occur mainly around site 2.…”

Section: Discussionmentioning

confidence: 99%

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

Szöllősi

Muallem

Csanády

et al. 2011

Journal of General Physiology

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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.

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Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity

Prins

Langron

Hastings

et al. 2019

Preprint

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18CFTR mutations cause cystic fibrosis by affecting how many channels reach the membrane, 19 and/or how well they work. This study presents an assay that simultaneously measures CFTR 20 biogenesis and function. A screen of 62 disease-causing mutations provides clues on how 21 approved drug VX-770 works.Abstract 24 Cystic fibrosis (CF) is a life-limiting disease caused by mutations in the CFTR gene, 25 encoding a plasma membrane anion-selective channel. Because CF-causing mutations affect 26 both CFTR permeation/gating and biogenesis, multi-assay approaches have been implemented 27 in drug development, sequentially screening for channel function and membrane density. Here 28 we present the first assay capable of simultaneous assessment of both CFTR characteristics. 29To validate our assay, we investigate F508del-CFTR, the most common disease-30 causing mutant, confirming rescue by incubation at low temperature, treatment with CFTR-31 targeting drugs and introduction of second-site revertant mutation R1070W. In addition, we 32 characterize a panel of 62 CF-causing mutations and profile effects of acute treatment with 33 approved drug VX-770 (ivacaftor). Measurements using the rare mutation panel correlate well 34 with published results, further validating the assay. Furthermore, mapping the potentiation 35 profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-36 770 might allow an open-channel conformation with an alternative arrangement of domain 37 interfaces around site 1. 38The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing 39 rapid and accurate inferences on gating/permeation properties and on how these are affected 40 by mutations and compounds. By providing a two-dimensional molecular characterization of 41 mutant CFTR proteins, our assay can better inform development of therapies tailored for 42 individual CFTR variants. Finally, the integrated assay boosts the potential for discovery of 43 dual-acting compounds, simultaneously repairing both biogenesis and function. 44 45 Keywords: protein transport, fluorescence imaging, precision medicine, VX-770.46 3 Non-standard Abbreviations 47 ABC ATP-binding cassette 48 CF Cystic Fibrosis 49 CFTR Cystic Fibrosis Transmembrane Conductance Regulator 50 FmCherry cell average normalized mCherry fluorescence intensity over the entire cell 51 FYFP cell average normalized YFP fluorescence intensity over the entire cell 52 FYFP membrane average normalized YFP fluorescence intensity within the membrane zone 53 GCFTR CFTR conductance 54 Gtrans transient anion conductance 55 IRES internal ribosome entry site 56 NBD nucleotide binding domain 57 PDL poly-D-lysine 58 PO open probability 59  CFTR membrane density, as defined in this paper 60 SSR sum of squared residuals 61 trans time constant of the transient anion conductance 62 TM transmembrane helix 63 VM membrane potential 64 WT wild type 65 YFP yellow fluorescent protein 66 67 Cystic fibrosis (CF) is a common life-limiting genetic disease. Alth...

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Extent of Nucleotide-Binding Domain (NBD) Separation When a CFTR Channel Closes

Cited by 4 publications

References 0 publications

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

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

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

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity

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