The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant

Deenah Osman,Cecilia Piergentili,Junjun Chen,Lucy N Sayer,Isabel Usón,Thomas G Huggins,Nigel J Robinson,Ehmke Pohl

doi:10.1074/jbc.m116.745174

Abstract

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

Highlights

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR
As observed in Salmonella [50], expression from PfrmRA-frmR was derepressed in the heterologous E. coli host following exposure of cells to maximum non-inhibitory concentrations (MNICs) of formaldehyde, whereas exposure to MNIC CoCl2 and ZnCl2 did not alleviate repression (Fig. 2E)
Detection of metals and formaldehyde by Salmonella FrmRE64H is retained when the sensor is expressed in a heterologous E. coli host (Fig. 2)

Summary

The Effectors of FrmR

Generation of a variant FrmR, responsive to cobalt and Zn(II) in addition to formaldehyde in vivo, is achieved by single amino acid substitution at the putative metal-binding site (Glu His) (Fig. 1). The intracellular effector of (any) FrmR is unknown, and possibilities include formaldehyde alone [1] or S-(hydroxymethyl)glutathione [2], in which case GSH could act negatively [3] or positively [4] on FrmR-mediated derepression. The FrmR sensory site is reactive to formaldehyde such that the related Salmonella RcnR sensor is less responsive to formaldehyde in vitro and in vivo. We determine the crystal structure of FrmRE64H to define the effectors and sensory site(s) of this formaldehyde- and metal-sensing variant. Implications for the basis of effector specificity within DUF156 family proteins and the chemical species detected by FrmR in vivo are discussed

Results

Introduction of the proposed formaldehyde sensing site of

Discussion

Experimental Procedures