Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity

Wong, Sharon L.; Awatade, Nikhil T; Astore, Miro A.; Allan, Katelin M.; Carnell, Michael; Šlapetová, Iveta; Chen, Po-Chia; Capraro, Alexander; Fawcett, Laura K.; Whan, Renée; Griffith, Renate; Ooi, Chee Y.; Kuyucak, Serdar; Jaffé, Adam; Waters, Shafagh A.

doi:10.1016/j.isci.2021.103710

Cited by 9 publications

(10 citation statements)

References 81 publications

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“…We next characterized the structural defect of R352Q-CFTR with molecular dynamics (MD) simulations, using an extended model of the phosphorylated, ATP-bound human CFTR ( 30 ). In line with the previously proposed role for R352 in forming a salt bridge ( 31 ), it was observed that mutation of the positively charged arginine (R) to neutral glutamine (Q) disrupted the R352-D993 salt bridge in the midsection of the CFTR channel pore ( Figure 4A , Figure E4).…”

Section: Resultsmentioning

confidence: 99%

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

Wong¹,

Awatade²,

Astore

et al. 2022

Am J Respir Cell Mol Biol

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A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

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Section: Resultsmentioning

confidence: 99%

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

Wong¹,

Awatade²,

Astore

et al. 2022

Am J Respir Cell Mol Biol

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“…Hence, our MD simulations have revealed a unique mode of pathogenesis for the S945L mutation that also provides a greater understanding of CFTR function. This is another example in a set of MD simulations of the CFTR protein which suggest that CFTR modulators can elicit a response in multiple CFTR missense mutations with unique molecular phenotypes (30)(31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

“…The CFTR protein model used for molecular dynamics simulations was based on the ATP-bound human CFTR cryogenic electron microscopy (cryo-EM) structure (PDB ID: 6MSM) ( 3 ). The construction of the CFTR protein system, included modelling part of the R domain, was described previously ( 31 ). In brief, the CFTR was embedded in a simplified cell membrane model containing pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids then solvated with 150 mM NaCl on both the cytoplasmic and periplasmic sides.…”

Section: Methodsmentioning

confidence: 99%

“…To decrease computational expense, elevated temperatures of 350 K (77°C) were used to accelerate conformational transitions, in addition to simulations at physiological temperatures of 310 K (37°C). The use of elevated temperatures to study defects in mutant CFTR was validated previously ( 31 ). All measurements of Root Mean Square Deviation (RMSD) were calculated using the positions of alpha carbons in the 6MSM PDB structure ( 3 ) as a reference.…”

Section: Methodsmentioning

confidence: 99%

“…This form of in vitro analysis has been permitted by the US Food and Drug Administration (FDA) to facilitate the approval of therapeutics for rare CFTR mutations in addition to in vitro testing systems using non-primary cells, such as Fisher Rat Thyroid (FRT) cells ( 21 ). CFTR-dependent chloride transport assays in patient-derived nasal epithelial cells ( 22 – 26 ) as well as forskolin-induced swelling assays in intestinal organoids ( 27 – 31 ) have been used in a patient-specific manner to predict modulator efficacy for the patient. This utilization of personalized cell models is particularly significant for those patients with rare or poorly characterized mutations that are unable to be tested in clinical trials.…”

Section: Introductionmentioning

confidence: 99%

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S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models

et al. 2022

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Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

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Novel gain-of-function mutants identify a critical region within CFTR membrane-spanning domain 2 controlling cAMP-dependent and ATP-independent channel activation

Castanier,

Elbahnsi,

Chevalier

et al. 2024

Cell. Mol. Life Sci.

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CFTR is an anion channel that has evolved from the mold of an ABC transporter. It possesses specific structural features, including a lateral portal between the cytoplasmic extensions of its transmembrane helices TM4 and TM6. This TM4-TM6 portal is lined by basic residues attracting anions from the cytosol towards the intracellular vestibule. Even though a symmetric, open portal is not observed at the level of the TM10/TM12 interface, basic amino acids are also present at this level, exposed to solvent in the vicinity of the regulatory R region, whose phosphorylation enables channel activation. Here, using all-atom molecular dynamics simulations in combination with functional and biochemical assays, we investigate the importance of these basic amino acids (R1158 and R1030), and of a neighboring aromatic amino acid (W846) in the regulation of CFTR activity. Results indicate that mutation of these amino acids globally increased channel activity and enabled channel opening by potentiators without the need to elevate cAMP levels. These effects (i) were observed even when the binding site of the potentiator VX-770 was mutated, revealing a probable independent mechanism, and (ii) were additive to one gain-of-function mutant within the selectivity filter. Taken together, our results indicate that the region of the membrane-spanning domain 2 (MSD2), symmetric to the lateral portal located between MSD1 TM4 and TM6, is a novel critical actor of CFTR regulation. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05431-9.

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Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity

Cited by 9 publications

References 81 publications

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models

Novel gain-of-function mutants identify a critical region within CFTR membrane-spanning domain 2 controlling cAMP-dependent and ATP-independent channel activation

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