Ligand-Controlled Proteolysis of theEscherichia coliTranscriptional Regulator ZntR

Abstract

Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntR R19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands-zinc and DNAfunction together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

High-Affinity Zn2+Uptake System ZnuABC Is Required for Bacterial Zinc Homeostasis in Intracellular Environments and Contributes to the Virulence ofSalmonella enterica

“…In fact, whereas the intracellular concentration of zinc in E. coli is in the millimolar range, Zur, the protein regulating the transcriptional activity of znuABC, and ZntR, the protein regulating the expression of the ZntA efflux pump, show femtomolar sensitivity to zinc, indicating that there is not a consistent pool of free zinc within the cell (37). Recent studies have also shown that ZntR and Zur are highly sensitive to extracellular zinc variations (51) and that the half-life of ZntR is enhanced when intracellular zinc levels are high (41), thus emphasizing the dynamic nature of intracellular zinc (10). Interestingly, a well-known feature of the acute-phase response which follows infection by gram-negative bacteria or the administration of lipopolysaccharide is a complex body redistribution of zinc; the plasmatic zinc concentration rapidly decreases, in association with its accumulation in liver and, to a lesser extent, in other tissues (21).…”

Clp-Dependent Proteolysis Down-Regulates Central Metabolic Pathways in Glucose-StarvedBacillus subtilis

“…It has been suggested that during the absence of substrates or cosubstrates a catalytic center or a metal binding site of a protein may be damaged; similarly, a protein can undergo structural perturbation without other components that normally hold it in native conformation (7,17,18,(31)(32)(33)41). These proteins could display a nonfunctional, unprotected, or even "unemployed" state, and it can be hypothesized that ATP-dependent proteases recognize these idle and defective proteins and degrade them.…”

A Galvanizing Story—Protein Stability and Zinc Homeostasis

“…The results of these studies can be explained in part by the new study by Pruteanu et al (15). Previously, this group dem-onstrated the role of the E. coli Clp proteases in controlling transcription factor levels under different growth conditions (4).…”

“…Less is known about the translational and posttranslational mechanisms of metal homeostasis in bacteria, although these are clearly important (9)(10)(11). The significance of protein stability and turnover is emphasized in the results of a study of the zinc-dependent ZntR transcriptional activator, which are presented in this issue (15). These new data provide a way to reconcile the results of previous biochemical and biological studies of ZntR function (6,13,18) and provide a better model for understanding zinc homeostasis and one which can be applied to other metals.…”