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 language | English (US) |
|---|---|
| Pages (from-to) | 17685-17704 |
| Number of pages | 20 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 46 |
| DOIs | |
| State | Published - Jan 1 2018 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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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 journal › Article
TY - JOUR
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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U2 - 10.1074/jbc.RA117.000819
DO - 10.1074/jbc.RA117.000819
M3 - Article
VL - 293
SP - 17685
EP - 17704
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 46
ER -