title: Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
abstract: Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.
authors.affiliation: From the Molecular Structure and Function Program, Hospital for Sick Children, and.
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Chong
authors.affiliation: From the Molecular Structure and Function Program, Hospital for Sick Children, and.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Farber
authors.affiliation: From the Molecular Structure and Function Program, Hospital for Sick Children, and.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Vernon
authors.affiliation: From the Molecular Structure and Function Program, Hospital for Sick Children, and.
authors.forename: Rhea P
authors.initials: RP
authors.lastname: Hudson
authors.affiliation: the Department of Chemistry, McGill University, Montreal, Quebec H3A 0B8, Canada.
authors.forename: Anthony K
authors.initials: AK
authors.lastname: Mittermaier
authors.affiliation: From the Molecular Structure and Function Program, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8 and forman@sickkids.ca.
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D000595:Amino Acid Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010649:Phenylalanine
keywords: ABC transporter; F508del; NBD dimerization; cystic fibrosis transmembrane conductance regulator (CFTR); dynamics; nuclear magnetic resonance (NMR); nucleotide-binding domain (NBD); protein processing; protein/protein interaction