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Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR

Mihaela Pruteanu,^1,2 Saskia B. Neher,^2, and Tania A. Baker^1,2*

Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,¹ Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139²

Received 1 October 2006/ Accepted 4 January 2007

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 DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

Published ahead of print on 12 January 2007.

Present address: UCSF/HHMI MC 2200, Genentech Hall N316, 600 16th Street, San Francisco, CA 94158-2517.

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