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A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution

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Abstract

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs–intracellular loop interaction in a way that would facilitate the open state of the channel.

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Acknowledgments

We thank Francesca Spanò and Olga Zegarra-Moran for their comments. This work was supported by the Italian Cystic Fibrosis Research Foundation Grant #7/2010, with the collaboration of Delegazione FFC di Cosenza 2, Work in Progress Communication “Sapore di Sale 2010”, Gruppo di Sostegno di Monterotondo (RM), Delegazione FFC di Genova, Delegazione FFC “Il Sorriso di Jenny”, LIFC Comitato provinciale di Livorno. We thank the ESRF for provision of synchrotron radiation facilities, and we would like to thank Petra Pernod for assistance in using beamline ID14-EH3.

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  1. Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini, 6, 16149, Genoa, Italy

    Elena Galfrè, Lauretta Galeno & Oscar Moran

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  1. Elena Galfrè
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  2. Lauretta Galeno
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Correspondence to Oscar Moran.

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Galfrè, E., Galeno, L. & Moran, O. A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution. Cell. Mol. Life Sci. 69, 3701–3713 (2012). https://doi.org/10.1007/s00018-012-1049-7

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