Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Multiple inherited mutations in the epithelial CFTR anion-permeable channel cause cystic fibrosis through different molecular mechanisms that can be targeted by different types of drugs to treat the disease. Drawing from available structural information and double-mutant cycle analysis of patch-clamp recordings, Simon and Csanády find that one of the most common CFTR disease-causing mutations, R117H, disrupts an interaction between the R117 side-chain and a main-chain carbonyl that selectively stabilizes the open state of the channel. These findings may open new paths of exploration for treating patients carrying this mutation, and provide important mechanistic constraints towards understanding the gating mechanism of CFTR proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Structural Biology and Molecular Biophysics (eLife)
Abstract
The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived ‘flickery’ nor in the long-lived ‘interburst’ closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.
Article activity feed
-
-
Evaluation Summary:
Multiple inherited mutations in the epithelial CFTR anion-permeable channel cause cystic fibrosis through different molecular mechanisms that can be targeted by different types of drugs to treat the disease. Drawing from available structural information and double-mutant cycle analysis of patch-clamp recordings, Simon and Csanády find that one of the most common CFTR disease-causing mutations, R117H, disrupts an interaction between the R117 side-chain and a main-chain carbonyl that selectively stabilizes the open state of the channel. These findings may open new paths of exploration for treating patients carrying this mutation, and provide important mechanistic constraints towards understanding the gating mechanism of CFTR proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
Multiple inherited mutations in the epithelial CFTR anion-permeable channel cause cystic fibrosis through different molecular mechanisms that can be targeted by different types of drugs to treat the disease. Drawing from available structural information and double-mutant cycle analysis of patch-clamp recordings, Simon and Csanády find that one of the most common CFTR disease-causing mutations, R117H, disrupts an interaction between the R117 side-chain and a main-chain carbonyl that selectively stabilizes the open state of the channel. These findings may open new paths of exploration for treating patients carrying this mutation, and provide important mechanistic constraints towards understanding the gating mechanism of CFTR proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
-
Reviewer #1 (Public Review):
Understanding the effects of cystic fibrosis-causing mutations in CFTR channel function, stability or expression is important because this determines the choice of treatment for the disease. The negative effects on gating of a common disease-causing mutation, R117H, were puzzling because it is located at an extracellular loop, away from the sites that control ATP-dependent gating in the channel. In the present manuscript, Simon and Csanády identify a hydrogen bond between the side chain of R117 and the main-chain carbonyl of E1124 that is present in a structure that is thought to closely represent the open state and absent in a structure representative of the closed state of the protein. The authors perform molecular modeling to identify that a residue deletion at E1124 is predicted to disrupt this …
Reviewer #1 (Public Review):
Understanding the effects of cystic fibrosis-causing mutations in CFTR channel function, stability or expression is important because this determines the choice of treatment for the disease. The negative effects on gating of a common disease-causing mutation, R117H, were puzzling because it is located at an extracellular loop, away from the sites that control ATP-dependent gating in the channel. In the present manuscript, Simon and Csanády identify a hydrogen bond between the side chain of R117 and the main-chain carbonyl of E1124 that is present in a structure that is thought to closely represent the open state and absent in a structure representative of the closed state of the protein. The authors perform molecular modeling to identify that a residue deletion at E1124 is predicted to disrupt this interaction, and show that CFTR channels with the deletion behave very similar to those carrying the single R117H mutation in regards to channel closure kinetics in a mutant background lacking ATP hydrolysis, consistent with the proposed interaction found in the structures. Using two different mutant backgrounds to disrupt ATP hydrolysis, and channels carrying either the R117H mutation or the E1224 deletion, or both perturbations, the authors measure the rates of channel opening and closure to both the resting state and a short-lived flickering closed state that occurs within open bursts of ATP-bound channels. From their measurements, the authors perform mutant cycle analysis and find that the two perturbations have non-additive effects consistent with a disruption of a stabilizing interaction that occurs only in the open state but not in the deactivated state or the short-lived closed state that occurs within open bursts. By comparing the predictions from kinetic models of channel function, the authors find that the energetics of disrupting the open state-stabilizing interaction can fully explain the major effects of the R117H mutation in the background channels utilized in the study, and suggest that a similar mechanism operates in WT CFTR channels carrying the R117H mutation. The data is of high quality, the analysis is carefully done, and the conclusions are well supported by the evidence that is provided, and are of both clinical and mechanistic relevance. Importantly, the finding the interaction established by R117H occurs only in the open state provides a relevant constraint for associating structures to specific functional states of the channel.
Although the conformational changes associated with formation or disruption of the interaction involving R117 are evident in the published structural models, it would be important to confirm whether these are supported by the experimental maps. Few details and data are provided in relation to the molecular dynamics simulations/molecular modeling that were carried out, which precludes evaluating the robustness of the calculations. The authors utilize the measurements in the D1370N background (Figure 3A) to calculate gating parameters from the kinetic models, but the burst-length in the R117H, E1124Δ, and R117H+E1124Δ appear to short in the recordings, raising concerns about the robustness of the parameters associated with the intra-burst transitions. Also regarding these intra-burst transitions, whereas the observed effects for the gating equilibrium constant are consistent with the authors' interpretation, the effects of the structural perturbations on the associated rate constants are intriguing: if the interaction occurs only in the open state, then the transition from the Cf state to the open state should not be affected by any of the perturbations, but this rate seems to also become altered, perhaps suggesting some degree of stabilization by the interaction in the Cf state or a destabilization of the transition state.
-
Reviewer #2 (Public Review):
Cystic Fibrosis (CF) is the most common fatal genetic disease in Caucasian populations. Disease-associated mutations of the CFTR gene often result in defects in opening/closing (or gating) of the CFTR channel. Recent breakthroughs in the development of drugs that target the CFTR protein itself pave the way for structure-based drug design, the success of which depends on our comprehensive understanding of how mutations cause functional abnormalities and how pharmaceutical reagents may act on CFTR channel folding and gating dynamics. Current studies by Simon and Csanady were meant to address the former by focusing on one mutation R117H commonly found in CF patients with less severe symptoms.
Major strengths of the manuscript include diligent utilization of the mutant cycle analysis, high-quality single-channel …
Reviewer #2 (Public Review):
Cystic Fibrosis (CF) is the most common fatal genetic disease in Caucasian populations. Disease-associated mutations of the CFTR gene often result in defects in opening/closing (or gating) of the CFTR channel. Recent breakthroughs in the development of drugs that target the CFTR protein itself pave the way for structure-based drug design, the success of which depends on our comprehensive understanding of how mutations cause functional abnormalities and how pharmaceutical reagents may act on CFTR channel folding and gating dynamics. Current studies by Simon and Csanady were meant to address the former by focusing on one mutation R117H commonly found in CF patients with less severe symptoms.
Major strengths of the manuscript include diligent utilization of the mutant cycle analysis, high-quality single-channel recordings and detailed data interpretations in the context of gating energetics.
This reviewer is more concerned with authors' structural interpretations of the data as there is no direct evidence for the assumed mutation-induced disruption of the hydrogen bond (e.g., E1124Δ) because it is the backbone carbonyl, not the side-chain, at position 1124 that is involved in hydrogen bond formation. Some molecular dynamic simulations were carried out to support this assumption, but the reported change of the hydrogen-bond distance by E1124Δ seems quite small. It is questionable if this change is adequate to explain quantitatively the reported 2.6 kT enthalpy change. Moreover, despite the fact that the hydrogen bond is found in the phosphorylated, ATP-bound structure of human CFTR, it is noted that this structure does not show a patent anion conduction pathway. Thus, some precautions are warranted when this structure is taken literally as the "open" channel conformation. Indeed, there are major discrepancies regarding pore-lining residues shown in this structure and those based on functional studies, suggesting that additional conformational changes in the transmembrane segments likely take place for the channel to sojourn to the true open state.
A few minor discrepancies between the current report and previous publications, although not necessarily affect their conclusions, may need clarification.
-
Reviewer #3 (Public Review):
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel crucial for salt and water transport across epithelial cells. CFTR mutations causes its dysfunction, and the dysfunction causes cystic fibrosis.
R117H is one of the most common mutations in cystic fibrosis. It was known that the R117H mutation affect ion channel gating and reduce conductance of the channel, but the molecular mechanism underlying is unclear. In this paper, the authors produced high-quality data through a very robust electrophysiology and thermodynamic approaches, and the data showed that a hydrogen bond between the arginine 117 side chain and the glutamate 1124 main chain carbonyl group on the extracellular side of CFTR stabilizes the open state of the ion channel. Therefore, the R117H mutation lowers the …
Reviewer #3 (Public Review):
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel crucial for salt and water transport across epithelial cells. CFTR mutations causes its dysfunction, and the dysfunction causes cystic fibrosis.
R117H is one of the most common mutations in cystic fibrosis. It was known that the R117H mutation affect ion channel gating and reduce conductance of the channel, but the molecular mechanism underlying is unclear. In this paper, the authors produced high-quality data through a very robust electrophysiology and thermodynamic approaches, and the data showed that a hydrogen bond between the arginine 117 side chain and the glutamate 1124 main chain carbonyl group on the extracellular side of CFTR stabilizes the open state of the ion channel. Therefore, the R117H mutation lowers the conductivity of the ion channel by breaking the hydrogen bond and induces a malfunction of CFTR.
There are five classes of cystic fibrosis mutations. By elucidating the molecular mechanism of these mutations, we can consider their application in therapeutics. Since the R117H mutation is a representative of Class IV CFTR mutations, which induce malfunction of ion conductance through the channel, researches on it, like presented in this paper, will guide the development of therapeutics targeting Class IV mutation.
-
