Effects of the cys mutations on structure and function of the ATP-dependent HslVU protease in Escherichia coli. The Cys287 to Val mutation in HslU uncouples the ATP-dependent proteolysis by HslvU from ATP hydrolysis.
To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
S J
SJ
Yoo
H H
HH
Kim
D H
DH
Shin
C S
CS
Lee
I S
IS
Seong
J H
JH
Seol
N
N
Shimbara
K
K
Tanaka
C H
CH
Chung
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D003545:Cysteine / Q000235:genetics*; D010450:Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D012694:Serine / Q000235:genetics; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014633:Valine / Q000235:genetics
D006360:Heat-Shock Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases; D014633:Valine; D003545:Cysteine; D005033:Ethylmaleimide; D000409:Alanine