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 A.
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 V.
AU - Riordan, John R.
AU - Brouillette, Christie G.
AU - Hunt, John F.
N1 - Funding Information:
This work was supported by grants from Cystic Fibrosis Foundation Thera-peutics Inc. to H. S., J. R. R., C. G. B., and J. F. H. as part of the CFTR 3D Struc-ture Consortium, including grant HUNT13XX0. The authors declare that they have no conflicts of interest with the contents of this article. The con-tent is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1–S8, Tables S1–S2, and supporting Refs. The atomic coordinates and structure factors (codes 5TF7, 5TF8, 5TFA, 5TFB, 5TFC, 5TFD, 5TFF, 5TFG, 5TGK, 5TFI, and 5TFJ) have been deposited in the Pro-tein Data Bank (http://wwpdb.org/). † Deceased, March 10, 2015. 1 Both authors contributed equally to this work. 2Present address: Dept. of Medicine Division of Hematology and Oncology, University of Alabama at Birmingham, Birmingham, AL 35294. 3 Supported by Grant 5U54GM094597 from the Protein Structure Initiative of the National Institutes of Health to the Northeast Structural Genomics Consortium. 4Present address: Irving Cancer Research Center, Columbia University, New York, NY 10032. 5Present address: Dept. of Neurosurgery and Department of Pharmacology, Pennsylvania State University, Hershey, PA 17033. 6 Present address: Laboratory of Cell and Developmental Signaling, NCI-Fred-erick, National Institutes of Health, Frederick, MD 21702. 7Supported by National Institutes of Health Training Grant T32GM008281 from the Training Program in Molecular Biophysics. 8Present address: Institut de Biologie Physico-chimique, CNRS UMR8261, Université Paris Diderot, Sorbonne Paris Cité, 13 Rue Pierre et Marie Curie, 75005 Paris, France. 9 Supported by National Institutes of Health Grant 1R35CA209896. 10Supported by National Institutes of Health Grants R01GM114015 and R01GM123247. Present address: Dept. of Pharmacology and Department of Biochemistry & Molecular Biology, Penn State College of Medicine, Her-shey, PA 17033. 11To whom correspondence should be addressed. Tel.: 212-854-5443; Fax: 212-865-8246; E-mail: jfh21@columbia.edu.
Funding Information:
Acknowledgments—We thank Cystic Fibrosis Foundation Therapeutics Inc. for long-term support of research by the CFTR 3D Structure Consortium, including the work reported in this paper. We thank Patrick Thibodeau and the other members of the CFTR 3D Structure Consortium for advice.
Funding Information:
This work was supported by grants from Cystic Fibrosis Foundation Therapeutics Inc. to H. S., J. R. R., C. G. B., and J. F. H. as part of the CFTR 3D Structure Consortium, including grant HUNT13XX0. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 3 Supported by Grant 5U54GM094597 from the Protein Structure Initiative of the National Institutes of Health to the Northeast Structural Genomics Consortium. 7 Supported by National Institutes of Health Training Grant T32GM008281 from the Training Program in Molecular Biophysics. 9 Supported by National Institutes of Health Grant 1R35CA209896. 10 Supported by National Institutes of Health Grants R01GM114015 and R01GM123247. We thank Cystic Fibrosis Foundation Therapeutics Inc. for long-term support of research by the CFTR 3D Structure Consortium, including the work reported in this paper. We thank Patrick Thibodeau and the other members of the CFTR 3D Structure Consortium for advice.
Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/11/16
Y1 - 2018/11/16
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
C2 - 29903914
AN - SCOPUS:85056594871
VL - 293
SP - 17685
EP - 17704
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 46
ER -