A structure-function study of ZraP and ZraS provides new insights into the two-component system Zra.

BACKGROUND:Zra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn²⁺, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. METHODS:The purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. RESULTS:We show that ZraS binds one Zn²⁺ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. CONCLUSION:Our results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. GENERAL SIGNIFICANCE:We bring new input on the so far poorly described Zra system and notably on ZraS.

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