PMID:17827297

Citation	Zhang, Y and Zuber, P (2007) Requirement of the zinc-binding domain of ClpX for Spx proteolysis in Bacillus subtilis and effects of disulfide stress on ClpXP activity. J. Bacteriol. 189:7669-80
Abstract	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.
Links	PubMed PMC2168722 Online version:10.1128/JB.00745-07
Keywords	Amino Acid Sequence; Bacillus subtilis/drug effects; Bacillus subtilis/genetics; Bacillus subtilis/metabolism; Bacterial Proteins/metabolism; Binding Sites; Diamide/pharmacology; Disulfides/pharmacology; Drug Stability; Endopeptidase Clp/metabolism; Genotype; Hydrogen Peroxide/pharmacology; Kinetics; Molecular Sequence Data; Transcription, Genetic; Zinc/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACSU:CLPX		GO:0030163: protein catabolic process	ECO:0000315:		P		Fig 1 and Fig 2	complete CACAO 6840
BACSU:CLPX	involved_in	GO:0030163: protein catabolic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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