title: Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR.
abstract: Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.
authors.affiliation: Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Aleksandrov
authors.forename: Pradeep
authors.initials: P
authors.lastname: Kota
authors.forename: Liying
authors.initials: L
authors.lastname: Cui
authors.forename: Tim
authors.initials: T
authors.lastname: Jensen
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Alekseev
authors.forename: Santiago
authors.initials: S
authors.lastname: Reyes
authors.forename: Lihua
authors.initials: L
authors.lastname: He
authors.forename: Martina
authors.initials: M
authors.lastname: Gentzsch
authors.forename: Luba A
authors.initials: LA
authors.lastname: Aleksandrov
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000495:Allosteric Site; D019943:Amino Acid Substitution; D000818:Animals; D001001:Anura; D002352:Carrier Proteins / Q000378:metabolism; D002461:Cell Line, Transformed; D002645:Chickens; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009711:Nucleotides / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D012754:Sharks; D012756:Sheep; D013816:Thermodynamics
chemical_list: C112303:CAVIN2 protein, human; C505032:CFTR protein, human; D002352:Carrier Proteins; D009711:Nucleotides; D028044:Phosphate-Binding Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011392:Proline