Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF1+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region.
The cystic fibrosis transmembrane conductance regulator (CFTR) is known to function as a regulated chloride channel and, when genetically impaired, to cause the disease cystic fibrosis. The novel studies reported here were undertaken to gain greater molecular insight into possible interactions among CFTR's soluble domains, which include two nucleotide binding domains (NBF1 and NBF2) and a regulatory domain (R). The NBF1+R and NBF2 regions of CFTR were highly expressed in Escherichia coli, purified to near homogeneity under denaturing conditions, and refolded. Both refolded proteins bound TNP-ATP and TNP-ADP, which could be readily replaced with ATP. Four different approaches were then used to determine whether the NBF1+R and NBF2 proteins interact. First, the purified NBF2 protein was labeled near its C-terminus with a fluorescent probe, 7-diethyl amino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Addition of the unlabeled NBF1+R to the CPM-labeled NBF2 caused a red-shift in lambda(max) of the CPM fluorescence, consistent with a direct interaction between the two proteins. Second, when the NBF1+R protein, the NBF2 protein, and a mixture of the two proteins were folded separately and analyzed by molecular sieve chomatography, the mixture was found to elute prior to either NBF1+R or NBF2. Third, na-tive-PAGE gel studies revealed that the mixture of the NBF1+R and NBF2 domains migrated as a single band with an R(F) value between that of NBF1+R and NBF2. Fourth, trypsin digestion of a mixture of the NBF1+R and NBF2 proteins occurred at a slower rate than that for the individual proteins. Finally, studies were carried out to determine whether an NBF1+R/NBF2 interaction could be demonstrated after expressing one of the two proteins in soluble, native form, thus avoiding the inclusion body, denaturation, and renaturation approach. Specifically, the NBF1+R protein was overexpressed in E. coli in fusion with glutathione-S-transferase near a thrombin cleavage site. Following binding of the GST-(NBF1+R) fusion protein to a GST Sepharose affinity column, added NBF2 was shown to bind and then to coelute with NBF1+R upon addition of glutathione or thrombin. Collectively, these experiments demonstrate that CFTR's NBF1+R region and its NBF2 domain, after folding separately as distinct units, have a strong propensity to interact and that this interaction is stable in the absence of added nucleotides or exogenously induced phosphorylation. These findings, together with the additional observation that the NBF1+R/NBF2 interaction induces a change in the C-terminus of NBF2, which resides within the C-terminal region of CFTR, may have important implications not only for the function of CFTR per se, but its interaction with other proteins.
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Department of Biological Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, Maryland, 21205-2185, USA.
Pedersen
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D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D003374:Coumarins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C505032:CFTR protein, human; D003374:Coumarins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D005982:Glutathione Transferase; D019791:Guanidine