Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore

Linsdell, Paul

doi:10.1007/5584_2016_33

Cited by 9 publications

(9 citation statements)

References 106 publications

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“…A current view is that NBD dimerization and dissociation transmit conformational changes via the ICLs to the transmembrane domains to gate the channel [32], [33], [34],69]. Interestingly, we observed large effects on T m func and T m trp by mutations in the Q-loop (S492P, S495P) thought to transmit signals to the transmembrane domains (for example 5SS, Table 1) [70]. Potentially, structural stabilization of NBD1 may also enhance these connections to the ICLs and so contribute to stabilization of the full-length CFTR protein.…”

Section: Discussionmentioning

confidence: 99%

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

Yang

Hildebrandt

Jiang

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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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 T > 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.

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Section: Discussionmentioning

confidence: 99%

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

Yang

Hildebrandt

Jiang

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The molecular mechanisms underlying the activation of CFTR channel function by PKA-dependent phosphorylation, ATP binding, and hydrolysis remain poorly understood mainly due to the paucity of structural information regarding the full-length protein. Hypothetical models for CFTR channel activation have been developed on the basis of patch clamp studies ( 35 – 37 ), cross-linking studies of cysteine-substituted CFTR in cellular membranes ( 8 , 10 , 38 ), and biophysical studies of isolated domains ( 4 , 7 ). In this work, the first spectroscopic studies of purified full-length CFTR together with cell-based studies of disease-causing mutants support the importance of interdomain interactions between the MSDs and NBDs in phosphorylation-dependent channel activation.…”

Section: Discussionmentioning

confidence: 99%

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface

Chin

Yang

Miles

et al. 2017

Journal of Biological Chemistry

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.

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“…MD simulations of ion channels have been used, for example, to explore the mechanism of ion permeation by external stimulants or to investigate the structural and functional effects of disease-causing mutations in the context of genetic disorders such as cystic fibrosis [385,386]. Such studies can give important insights to aid in the drug discovery process.…”

Section: Molecular Dynamics Simulations Of Gpcrs and Ion Channelsmentioning

confidence: 99%

Molecular Dynamics Simulations in Drug Discovery and Pharmaceutical Development

et al. 2020

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Molecular dynamics (MD) simulations have become increasingly useful in the modern drug development process. In this review, we give a broad overview of the current application possibilities of MD in drug discovery and pharmaceutical development. Starting from the target validation step of the drug development process, we give several examples of how MD studies can give important insights into the dynamics and function of identified drug targets such as sirtuins, RAS proteins, or intrinsically disordered proteins. The role of MD in antibody design is also reviewed. In the lead discovery and lead optimization phases, MD facilitates the evaluation of the binding energetics and kinetics of the ligand-receptor interactions, therefore guiding the choice of the best candidate molecules for further development. The importance of considering the biological lipid bilayer environment in the MD simulations of membrane proteins is also discussed, using G-protein coupled receptors and ion channels as well as the drug-metabolizing cytochrome P450 enzymes as relevant examples. Lastly, we discuss the emerging role of MD simulations in facilitating the pharmaceutical formulation development of drugs and candidate drugs. Specifically, we look at how MD can be used in studying the crystalline and amorphous solids, the stability of amorphous drug or drug-polymer formulations, and drug solubility. Moreover, since nanoparticle drug formulations are of great interest in the field of drug delivery research, different applications of nano-particle simulations are also briefly summarized using multiple recent studies as examples. In the future, the role of MD simulations in facilitating the drug development process is likely to grow substantially with the increasing computer power and advancements in the development of force fields and enhanced MD methodologies.

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Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore

Cited by 9 publications

References 106 publications

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

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

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface

Molecular Dynamics Simulations in Drug Discovery and Pharmaceutical Development

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