Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants.
The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 degrees C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s(-1), respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518-638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s(-1). These results show that the loss of the lid does not interfere with interdomain communication, that the beta-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK.peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK.peptide complex.
Buczynski
G
G
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
Slepenkov
S V
SV
Sehorn
M G
MG
Witt
S N
SN
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli