Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization

Mihályi, Csaba; Törőcsik, Beáta; Csanády, László

doi:10.7554/elife.18164

Cited by 27 publications

(26 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Instead, the transition between opened and closed NBDs – and consequently – the transition between the IF and OF state likely occurs spontaneously by Brownian motion even in the absence of nucleotides. This was demonstrated previously by cross-linking experiments on TM287/288 (Hohl et al, 2012), cryo-EM studies on MsbA (Moeller et al, 2015) and for CFTR, which opens its pore spontaneously at low frequency even in the absence of nucleotides (Mihályi et al, 2016). To stabilize the closed NBD dimer by two sandwiched nucleotides resulting in an OF orientation of the TMDs (state 2 in Figure 10), a second nucleotide has to bind to the consensus site (state 1b) during the process of NBD closure.…”

Section: Discussionsupporting

confidence: 67%

Exploring conformational equilibria of a heterodimeric ABC transporter

Timachi

Hutter

Höhl

et al. 2017

eLife

View full text Add to dashboard Cite

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.DOI: http://dx.doi.org/10.7554/eLife.20236.001

show abstract

Section: Discussionsupporting

confidence: 67%

Exploring conformational equilibria of a heterodimeric ABC transporter

Timachi

Hutter

Höhl

et al. 2017

eLife

View full text Add to dashboard Cite

show abstract

“…Another interesting case where cytoplasmic regions control transport functioning directly occurs in prokaryotic ATPbinding cassette (ABC) transporters or the homologous human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel involved in cystic fibrosis [Gadsby et al 2006;Mihályi et al 2016, for a review see Locher (2016)]. In particular, in CFTR and other ABC transporters, ATP-bindingdependent dimerization of two cytosolic nucleotide-binding domains (NBDs) opens the pore, whereas dimer disruption following ATP hydrolysis closes it.…”

Section: Discussionmentioning

confidence: 99%

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions

2017

View full text Add to dashboard Cite

FurE, a member of the Nucleobase Cation Symporter 1 transporter family in , is specific for allantoin, uric acid (UA), uracil, and related analogs. Herein, we show that C- or N-terminally-truncated FurE transporters (FurE-ΔC or FurE-ΔΝ) present increased protein stability, but also an inability for UA transport. To better understand the role of cytoplasmic terminal regions, we characterized genetic suppressors that restore FurE-ΔC-mediated UA transport. Suppressors map in the periphery of the substrate-binding site [Thr133 in transmembrane segment (TMS)3 and Val343 in TMS8], an outward-facing gate (Ser296 in TMS7, Ile371 in TMS9, and Tyr392 and Leu394 in TMS10), or in flexible loops (Asp26 in L, Gly222 in L5, and Asn308 in L7). Selected suppressors were also shown to restore the wild-type specificity of FurE-ΔΝ, suggesting that both C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection. A direct, substrate-sensitive interaction of C- and/or N-terminal domains was supported by bimolecular fluorescence complementation assays. To our knowledge, this is the first case where not only the function, but also the specificity, of a eukaryotic transporter is regulated by its terminal cytoplasmic regions.

show abstract

“…Phosphorylated WT CFTR channels open infrequently even in the absence of ATP ( Figure 6B , top current trace ), and such ‘spontaneous’ openings reflect occasional dimerization of empty, unliganded NBDs ( Mihályi et al, 2016 ) ( Figure 6B , top cartoon ). Comparison of such spontaneous gating of WT channels with the equilibrium gating of a hydrolysis-deficient mutant, such as the NBD2 Walker A lysine mutant K1250R, in saturating ATP ( Figure 6B , bottom current trace ) reveals two robust effects of bound ATP: a shortening of closed interburst durations ( Figure 6B , compare green bars above current traces) and a lengthening of open burst durations ( Figure 6B , compare red bars below current traces), both by >100 fold ( Mihályi et al, 2016 ). Thus, bound ATP reduces the energetic barrier for opening, but increases the barrier for non-hydrolytic closure ( Figure 6C , black vs. orange standard Gibbs energy profiles).…”

Section: Discussionmentioning

confidence: 99%

Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions

Sorum

Törőcsik

Csanády

2017

eLife

Self Cite

View full text Add to dashboard Cite

CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, is opened by ATP binding to two cytosolic nucleotide binding domains (NBDs), but pore-domain mutations may also impair gating. ATP-bound NBDs dimerize occluding two nucleotides at interfacial binding sites; one site hydrolyzes ATP, the other is inactive. The pore opens upon tightening, and closes upon disengagement, of the catalytic site following ATP hydrolysis. Extent, timing, and role of non-catalytic-site movements are unknown. Here we exploit equilibrium gating of a hydrolysis-deficient mutant and apply Φ value analysis to compare timing of opening-associated movements at multiple locations, from the cytoplasmic ATP sites to the extracellular surface. Marked asynchrony of motion in the two ATP sites reveals their distinct roles in channel gating. The results clarify the molecular mechanisms of functional cross-talk between canonical and degenerate ATP sites in asymmetric ABC proteins, and of the gating defects caused by two common CF mutations.

show abstract

Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization

Cited by 27 publications

References 25 publications

Exploring conformational equilibria of a heterodimeric ABC transporter

Exploring conformational equilibria of a heterodimeric ABC transporter

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions

Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions

Contact Info

Product

Resources

About