Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli.
HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.
Shin
D H
DH
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, South Korea.
Yoo
S J
SJ
Shim
Y K
YK
Seol
J H
JH
Kang
M S
MS
Chung
C H
CH
D049069:ATP-Dependent Proteases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D049071:Endopeptidase Clp; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006360:Heat-Shock Proteins; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011108:Polymers; D012697:Serine Endopeptidases / Q000378:metabolism
D006360:Heat-Shock Proteins; D009842:Oligopeptides; D011108:Polymers; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases