Requirement of the zinc-binding domain of ClpX for Spx proteolysis in Bacillus subtilis and effects of disulfide stress on ClpXP activity.
Spx, a transcriptional regulator of the disulfide stress response in Bacillus subtilis, is under the proteolytic control of the ATP-dependent protease ClpXP. Previous studies suggested that ClpXP activity is down-regulated in response to disulfide stress, resulting in elevated concentrations of Spx. The effect of disulfide stress on ClpXP activity was examined using the thiol-specific oxidant diamide. ClpXP-catalyzed degradation of either Spx or a green fluorescent protein derivative bearing an SsrA tag recognized by ClpXP was inhibited by diamide treatment in vitro. Spx is also a substrate for MecA/ClpCP-catalyzed proteolysis in vitro, but diamide used at the concentrations that inhibited ClpXP had little observable effect on MecA/ClpCP activity. ClpX bears a Cys4 Zn-binding domain (ZBD), which in other Zn-binding proteins is vulnerable to thiol-reactive electrophiles. Diamide treatment caused partial release of Zn from ClpX and the formation of high-molecular-weight species, as observed by electrophoresis through nonreducing gels. Reduced Spx proteolysis in vitro and elevated Spx concentration in vivo resulted when two of the Zn-coordinating Cys residues of the ClpX ZBD were changed to Ser. This was reflected in enhanced Spx activity in both transcription activation and repression in cells expressing the Cys-to-Ser mutants. ClpXP activity in vivo is reduced when cells are exposed to diamide, as shown by the enhanced stability of an SsrA-tagged protein after treatment with the oxidant. The results are consistent with the hypothesis that inhibition of ClpXP by disulfide stress is due to structural changes to the N-terminal ZBD of ClpX.
Zhang
Ying
Y
Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Rd., Beaverton, OR 97006, USA.
Zuber
Peter
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology*; D004355:Drug Stability; D049071:Endopeptidase Clp / Q000378:metabolism*; D005838:Genotype; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism