DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Increases in cytosolic Ca2+ induce dynamin- and calcineurin-dependent internalisation of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca2+. However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.

Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator.

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

Transmembrane helical interactions in the CFTR channel pore.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF). Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF) and develop an inward (IWF) facing model employing an integrated experimental-molecular dynamics simulation (200 ns) approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs) of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 °C and remain monodisperse at 4 °C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

Rational Coupled Dynamics Network Manipulation Rescues Disease-Relevant Mutant Cystic Fibrosis Transmembrane Conductance Regulator.

Many cellular functions necessary for life are tightly regulated by protein allosteric conformational change, and correlated dynamics between protein regions has been found to contribute to the function of proteins not previously considered allosteric. The ability to map and control such dynamic coupling would thus create opportunities for the extension of current therapeutic design strategy. Here, we present an approach to determine the networks of residues involved in the transfer of correlated motion across a protein, and apply our approach to rescue disease-causative mutant cystic fibrosis transmembrane regulator (CFTR) ion channels, ΔF508 and ΔI507, which together constitute over 90% of cystic fibrosis cases. We show that these mutations perturb dynamic coupling within the first nucleotide-binding domain (NBD1), and uncover a critical residue that mediates trans-domain coupled dynamics. By rationally designing a mutation to this residue, we improve aberrant dynamics of mutant CFTR as well as enhance surface expression and function of both mutants, demonstrating the rescue of a disease mutation by rational correction of aberrant protein dynamics.

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 µg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

Restoration of NBD1 thermal stability is necessary and sufficient to correct ∆F508 CFTR folding and assembly.

Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ∆F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR.

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration.

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.

Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

Relationship between nucleotide binding and ion channel gating in cystic fibrosis transmembrane conductance regulator.

We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and Phi values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional Phi values were not observed in either of these distant sites suggests that there may not be a deterministic 'lever-arm' mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel.

Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.