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

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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.

No MeSH data available.


Conservation of residues in the DUF156 FrmR subgroup and proposed formaldehyde-sensing site.A, alignment of Salmonella FrmR with nonredundant UniProtKB DUF156 sequences previously attributed to the FrmR subgroup (45). Organism details and UniProtKB identifiers are outlined under “Experimental Procedures.” Highlighted in gray are residues conserved in both FrmR and RcnR subgroups. Highlighted in red are residues conserved in the FrmR but not RcnR subgroup. Highlighted in yellow is the invariant cysteine present in all DUF156 proteins. The secondary structure elements of the FrmRE64H crystal structure are shown below (black bars). The inferred Zn(II)/Co(II)-sensing site is identified by orange arrows. The proposed formaldehyde sensing site is identified by green arrows. B and C, dimeric representation of FrmRE64H with the side chains for Cys35 and FrmR subgroup-specific residues labeled. Each monomer is colored differently (using the same colors as in Fig. 3A) with secondary structure units labeled on the cyan subunit. D, solvent-accessible surface representation of the proposed formaldehyde-binding site, which comprises Pro2 (subunit 1, cyan) and Cys35 (subunit 2, green). E, proposed reaction of formaldehyde with FrmR Cys35 (green) followed by Pro2 (cyan) (both deprotonated ultimately to water) via an S-hydroxymethyl intermediate. The reciprocal reaction with Pro2 followed by Cys35 via an N-methylol intermediate is also possible. In both cases, a methylene bridge (black) between the two residues is the final product. The nucleophile(s) responsible for deprotonation of Cys35 and Pro2 remain unknown.
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Figure 5: Conservation of residues in the DUF156 FrmR subgroup and proposed formaldehyde-sensing site.A, alignment of Salmonella FrmR with nonredundant UniProtKB DUF156 sequences previously attributed to the FrmR subgroup (45). Organism details and UniProtKB identifiers are outlined under “Experimental Procedures.” Highlighted in gray are residues conserved in both FrmR and RcnR subgroups. Highlighted in red are residues conserved in the FrmR but not RcnR subgroup. Highlighted in yellow is the invariant cysteine present in all DUF156 proteins. The secondary structure elements of the FrmRE64H crystal structure are shown below (black bars). The inferred Zn(II)/Co(II)-sensing site is identified by orange arrows. The proposed formaldehyde sensing site is identified by green arrows. B and C, dimeric representation of FrmRE64H with the side chains for Cys35 and FrmR subgroup-specific residues labeled. Each monomer is colored differently (using the same colors as in Fig. 3A) with secondary structure units labeled on the cyan subunit. D, solvent-accessible surface representation of the proposed formaldehyde-binding site, which comprises Pro2 (subunit 1, cyan) and Cys35 (subunit 2, green). E, proposed reaction of formaldehyde with FrmR Cys35 (green) followed by Pro2 (cyan) (both deprotonated ultimately to water) via an S-hydroxymethyl intermediate. The reciprocal reaction with Pro2 followed by Cys35 via an N-methylol intermediate is also possible. In both cases, a methylene bridge (black) between the two residues is the final product. The nucleophile(s) responsible for deprotonation of Cys35 and Pro2 remain unknown.

Mentions: To define the functional formaldehyde sensory site, residues specifically conserved within the FrmR subgroup of the DUF156 family of transcriptional regulators were identified. Protein sequences previously ascribed to the FrmR subgroup (45) were used to generate a multiple-sequence alignment with Salmonella FrmR (Fig. 5A). Twelve residues are conserved within the FrmR subgroup but absent from the closely related Ni(II)/Co(II)-sensing RcnR subgroup. Two-thirds of the conserved residues are clustered in helix α1 based on the structure of FrmRE64H (Fig. 5, A–C). Sensing of formaldehyde may proceed via reaction with Cys35, also implicated in the FrmR metal site (Fig. 4, D and E) due to its conservation in all characterized DUF156 members. Formation of an S-formyl adduct at this Cys-thiol followed by reaction with a primary amine has been suggested as a possible reaction mechanism of FrmR with formaldehyde (30). The pyrrolidine side chain of proline residue 2 (α1) is in close proximity (3.0–3.2 Å in the four independent locations within the tetrameric structure) to Cys35 from α2′ (Fig. 5, B and C, and supplemental Fig. S1A). A second FrmR-specific proline (Pro5) acts to terminate helix α1 and positions the amino terminus of FrmRE64H adjacent to Cys35 (Fig. 5B). Pro2 is the first residue identified in the FrmRE64H structure and is positioned in a pocket at the dimer interface, leaving no space (and no observed electron density) for the amino-terminal methionine predicted by the primary sequence (Fig. 5D and supplemental Fig. S1A). The amino-terminal region has been implicated in the coordination of Ni(II)/Co(II) by RcnR and of Ni(II) by InrS (61, 62). In the absence of Met1, the terminal secondary amine of Pro2 and a Cys35-thiolate are both ideal candidates for nucleophilic addition to formaldehyde (Fig. 5, D and E) (63, 64). Either reaction with Pro2 followed by Cys35 via an N-methylol intermediate or reciprocally via an S-hydroxymethyl intermediate is plausible (Fig. 5E). In both cases, the end product would be a methylene bridge between the two residues, requiring a 1:1 formaldehyde/FrmR monomer (4 possible sites/tetramer) reaction stoichiometry.


The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *
Conservation of residues in the DUF156 FrmR subgroup and proposed formaldehyde-sensing site.A, alignment of Salmonella FrmR with nonredundant UniProtKB DUF156 sequences previously attributed to the FrmR subgroup (45). Organism details and UniProtKB identifiers are outlined under “Experimental Procedures.” Highlighted in gray are residues conserved in both FrmR and RcnR subgroups. Highlighted in red are residues conserved in the FrmR but not RcnR subgroup. Highlighted in yellow is the invariant cysteine present in all DUF156 proteins. The secondary structure elements of the FrmRE64H crystal structure are shown below (black bars). The inferred Zn(II)/Co(II)-sensing site is identified by orange arrows. The proposed formaldehyde sensing site is identified by green arrows. B and C, dimeric representation of FrmRE64H with the side chains for Cys35 and FrmR subgroup-specific residues labeled. Each monomer is colored differently (using the same colors as in Fig. 3A) with secondary structure units labeled on the cyan subunit. D, solvent-accessible surface representation of the proposed formaldehyde-binding site, which comprises Pro2 (subunit 1, cyan) and Cys35 (subunit 2, green). E, proposed reaction of formaldehyde with FrmR Cys35 (green) followed by Pro2 (cyan) (both deprotonated ultimately to water) via an S-hydroxymethyl intermediate. The reciprocal reaction with Pro2 followed by Cys35 via an N-methylol intermediate is also possible. In both cases, a methylene bridge (black) between the two residues is the final product. The nucleophile(s) responsible for deprotonation of Cys35 and Pro2 remain unknown.
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Figure 5: Conservation of residues in the DUF156 FrmR subgroup and proposed formaldehyde-sensing site.A, alignment of Salmonella FrmR with nonredundant UniProtKB DUF156 sequences previously attributed to the FrmR subgroup (45). Organism details and UniProtKB identifiers are outlined under “Experimental Procedures.” Highlighted in gray are residues conserved in both FrmR and RcnR subgroups. Highlighted in red are residues conserved in the FrmR but not RcnR subgroup. Highlighted in yellow is the invariant cysteine present in all DUF156 proteins. The secondary structure elements of the FrmRE64H crystal structure are shown below (black bars). The inferred Zn(II)/Co(II)-sensing site is identified by orange arrows. The proposed formaldehyde sensing site is identified by green arrows. B and C, dimeric representation of FrmRE64H with the side chains for Cys35 and FrmR subgroup-specific residues labeled. Each monomer is colored differently (using the same colors as in Fig. 3A) with secondary structure units labeled on the cyan subunit. D, solvent-accessible surface representation of the proposed formaldehyde-binding site, which comprises Pro2 (subunit 1, cyan) and Cys35 (subunit 2, green). E, proposed reaction of formaldehyde with FrmR Cys35 (green) followed by Pro2 (cyan) (both deprotonated ultimately to water) via an S-hydroxymethyl intermediate. The reciprocal reaction with Pro2 followed by Cys35 via an N-methylol intermediate is also possible. In both cases, a methylene bridge (black) between the two residues is the final product. The nucleophile(s) responsible for deprotonation of Cys35 and Pro2 remain unknown.
Mentions: To define the functional formaldehyde sensory site, residues specifically conserved within the FrmR subgroup of the DUF156 family of transcriptional regulators were identified. Protein sequences previously ascribed to the FrmR subgroup (45) were used to generate a multiple-sequence alignment with Salmonella FrmR (Fig. 5A). Twelve residues are conserved within the FrmR subgroup but absent from the closely related Ni(II)/Co(II)-sensing RcnR subgroup. Two-thirds of the conserved residues are clustered in helix α1 based on the structure of FrmRE64H (Fig. 5, A–C). Sensing of formaldehyde may proceed via reaction with Cys35, also implicated in the FrmR metal site (Fig. 4, D and E) due to its conservation in all characterized DUF156 members. Formation of an S-formyl adduct at this Cys-thiol followed by reaction with a primary amine has been suggested as a possible reaction mechanism of FrmR with formaldehyde (30). The pyrrolidine side chain of proline residue 2 (α1) is in close proximity (3.0–3.2 Å in the four independent locations within the tetrameric structure) to Cys35 from α2′ (Fig. 5, B and C, and supplemental Fig. S1A). A second FrmR-specific proline (Pro5) acts to terminate helix α1 and positions the amino terminus of FrmRE64H adjacent to Cys35 (Fig. 5B). Pro2 is the first residue identified in the FrmRE64H structure and is positioned in a pocket at the dimer interface, leaving no space (and no observed electron density) for the amino-terminal methionine predicted by the primary sequence (Fig. 5D and supplemental Fig. S1A). The amino-terminal region has been implicated in the coordination of Ni(II)/Co(II) by RcnR and of Ni(II) by InrS (61, 62). In the absence of Met1, the terminal secondary amine of Pro2 and a Cys35-thiolate are both ideal candidates for nucleophilic addition to formaldehyde (Fig. 5, D and E) (63, 64). Either reaction with Pro2 followed by Cys35 via an N-methylol intermediate or reciprocally via an S-hydroxymethyl intermediate is plausible (Fig. 5E). In both cases, the end product would be a methylene bridge between the two residues, requiring a 1:1 formaldehyde/FrmR monomer (4 possible sites/tetramer) reaction stoichiometry.

View Article: PubMed Central - PubMed

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.

No MeSH data available.