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CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

Nature Structural & Molecular Biology volume 14, pages 738–745 (2007)Cite this article

Abstract

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.

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Figure 1: R-region phosphorylation.
Figure 2: Structural properties of the free R region.
Figure 3: Interaction of the R region with NBD1.
Figure 4: Analysis of R-region interactions with NBD1.
Figure 5: Schematic illustrating how phosphorylation-induced structural changes in the R region lead to a redistribution of binding equilibria with various regulatory interaction partners.

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Acknowledgements

We thank R. Muhandiram and L.E. Kay for technical assistance with NMR experiments, D.F. Hansen for assistance with NMR data analysis using Fuda, C.E. Bear and T. Mittag for many helpful discussions, and J.W. Hanrahan for critically reading the manuscript. This work was funded by grants from the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research to J.D.F.-K. and from the US National Institutes of Health (DK49835) and the Robert Welch Foundation to P.J.T. J.M.R.B. was supported by scholarships from the Natural Sciences and Engineering Research Council of Canada.

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  1. Wing-Yiu Choy

    Present address: Present address: Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.,

Authors and Affiliations

  1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Ave., Toronto, M5G 1X8, Ontario, Canada

    Jennifer M R Baker, Rhea P Hudson, Voula Kanelis & Julie D Forman-Kay

  2. Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Jennifer M R Baker, Wing-Yiu Choy & Julie D Forman-Kay

  3. Department of Physiology, 6001 Forest Park Lane, University of Texas Southwestern Medical Center, Dallas, 75390-9040, Texas, USA

    Patrick H Thibodeau & Philip J Thomas

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Contributions

J.M.R.B. designed experiments, expressed and purified R region, performed NMR experiments, analyzed data and wrote the manuscript; R.P.H. expressed and purified NBD1; V.K. analyzed data; W.-Y.C. analyzed NMR data; P.H.T. generated the NBD1 construct and advised on NBD1 purification; P.J.T. contributed new reagents and analyzed data; J.D.F.-K. directed the study, designed experiments and wrote the manuscript. All the authors edited the manuscript.

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Correspondence to Julie D Forman-Kay.

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The authors declare no competing financial interests.

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Baker, J., Hudson, R., Kanelis, V. et al. CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices. Nat Struct Mol Biol 14, 738–745 (2007). https://doi.org/10.1038/nsmb1278

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