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

Chi Wang, Andrei A. Aleksandrov, Zhengrong Yang, Farhad Forouhar, Elizabeth Anne Proctor, Pradeep Kota, Jianli An, Anna Kaplan, Netaly Khazanov, Grégory Boël, Brent R. Stockwell, Hanoch Senderowitz, Nikolay Dokholyan, John R. Riordan, Christie G. Brouillette, John F. Hunt

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)17685-17704
Number of pages20
JournalJournal of Biological Chemistry
Volume293
Issue number46
DOIs
StatePublished - Jan 1 2018

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Cystic Fibrosis Transmembrane Conductance Regulator
Ligands
Mutation
Nucleotides
Molecules
Conformations
Agglomeration
Stabilization
Protein Conformation
Protein Stability
Quenching
Assays
Thermodynamics
Proteins
Adenosine Triphosphate
Fluorescence
Temperature

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Wang, Chi ; Aleksandrov, Andrei A. ; Yang, Zhengrong ; Forouhar, Farhad ; Proctor, Elizabeth Anne ; Kota, Pradeep ; An, Jianli ; Kaplan, Anna ; Khazanov, Netaly ; Boël, Grégory ; Stockwell, Brent R. ; Senderowitz, Hanoch ; Dokholyan, Nikolay ; Riordan, John R. ; Brouillette, Christie G. ; Hunt, John F. / Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 46. pp. 17685-17704.
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abstract = "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.",
author = "Chi Wang and Aleksandrov, {Andrei A.} and Zhengrong Yang and Farhad Forouhar and Proctor, {Elizabeth Anne} and Pradeep Kota and Jianli An and Anna Kaplan and Netaly Khazanov and Gr{\'e}gory Bo{\"e}l and Stockwell, {Brent R.} and Hanoch Senderowitz and Nikolay Dokholyan and Riordan, {John R.} and Brouillette, {Christie G.} and Hunt, {John F.}",
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Wang, C, Aleksandrov, AA, Yang, Z, Forouhar, F, Proctor, EA, Kota, P, An, J, Kaplan, A, Khazanov, N, Boël, G, Stockwell, BR, Senderowitz, H, Dokholyan, N, Riordan, JR, Brouillette, CG & Hunt, JF 2018, 'Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator', Journal of Biological Chemistry, vol. 293, no. 46, pp. 17685-17704. https://doi.org/10.1074/jbc.RA117.000819

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator. / Wang, Chi; Aleksandrov, Andrei A.; Yang, Zhengrong; Forouhar, Farhad; Proctor, Elizabeth Anne; Kota, Pradeep; An, Jianli; Kaplan, Anna; Khazanov, Netaly; Boël, Grégory; Stockwell, Brent R.; Senderowitz, Hanoch; Dokholyan, Nikolay; Riordan, John R.; Brouillette, Christie G.; Hunt, John F.

In: Journal of Biological Chemistry, Vol. 293, No. 46, 01.01.2018, p. 17685-17704.

Research output: Contribution to journalArticle

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T1 - Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator

AU - Wang, Chi

AU - Aleksandrov, Andrei A.

AU - Yang, Zhengrong

AU - Forouhar, Farhad

AU - Proctor, Elizabeth Anne

AU - Kota, Pradeep

AU - An, Jianli

AU - Kaplan, Anna

AU - Khazanov, Netaly

AU - Boël, Grégory

AU - Stockwell, Brent R.

AU - Senderowitz, Hanoch

AU - Dokholyan, Nikolay

AU - Riordan, John R.

AU - Brouillette, Christie G.

AU - Hunt, John F.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

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