Abstract
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.
Highlights
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
NEM 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. Either mutation abolished both the basal and HslUactivated 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
Effects of the Cys Mutations on the Ability of HslU in ATP Hydrolysis and in Supporting HslV-mediated Proteolysis—In order to determine the effects of Cys mutations on the ATPase activity of HslU, site-directed mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val (Fig. 1)
Summary
Carbobenzoxy; AMC, 7-amido-4methyl coumarin; NEM, N-ethylmaleimide; HPLC, high pressure liquid chromatography. It has recently been revealed that ,␥-imido-ATP and ATP␥S, both of which are nonhydrolyzable ATP analogs, can support the proteolytic activity of the HslVU protease [33, 34]. These findings suggest that proteolysis by HslVU can be uncoupled from ATP cleavage. In order to define the role of the Cys residues of HslU in ATP hydrolysis and to clarify further the inhibitory effect of NEM, site-directed mutagenesis was carried out to replace either the 261st or the 287th residue (Cys) in HslU with Val. We replaced the 159th residue (Cys) in HslV with Ser or Ala to examine the effects of the mutations on the proteolytic function of the HslVU protease. We determined the cleavage specificity of the enzyme using insulin B-chain as a model substrate
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