The molecular chaperone Hsc70 assists the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator.
The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator is a single amino acid deletion (DeltaF508) in the N-terminal cytosolic nucleotide-binding domain (NBD1). This mutation has previously been shown to be a temperature-sensitive folding mutation that alters the folding pathway but not the native state stability of the isolated domain (Qu, B.-H., and Thomas, P. J. (1996) J. Biol. Chem. 271, 7261-7264). Here we provide evidence that the molecular chaperone Hsc70 productively interacts with NBD1 to increase the folding yield of the domain and inhibit off-pathway associations leading to the formation of high molecular weight aggregates. Furthermore, we have sublocalized a region within NBD1 where Hsc70 binds. Notably, inhibition of NBD1 aggregation is not dependent upon the presence of Hsc70 in the early stages of folding, indicating that the chaperone may act on a folding intermediate. In the presence of K+ and Mg2+-ATP, conditions where Hsp70 binds substrate rapidly and can release it, Hsc70 is less effective at inhibiting NBD1 aggregation. Thus, the rate of release of unfolded substrate is an important factor in preventing aggregation and promoting folding of the domain. These results demonstrate that Hsc70 promotes the otherwise inefficient folding of DeltaF-NBD1 and provide insight into the mechanisms by which molecular chaperones assist proteins in folding.
Strickland
E
E
Graduate Program in Molecular Biophysics, the University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040, USA.
Qu
B H
BH
Millen
L
L
Thomas
P J
PJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D017510:Protein Folding
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate