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Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

Lou YC, Weng TH, Li YC, Kao YF, Lin WF, Peng HL, Chou SH, Hsiao CD, Chen C - Nat Commun (2015)

Bottom Line: However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution.Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression.We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.

ABSTRACT
PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

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Related in: MedlinePlus

NMR investigations of the PmrA–DNA complex.The overlaid two-dimensional (2D) 1H, 15N TROSY-HSQC spectra (a) and 2D 1H, 13C HSQC spectra, showing the methyl resonances of Ile (b), Leu and Val (c), acquired from PmrA in the absence (black) and presence of DNA (red). The residues that show two sets of amide or methyl resonances in the DNA-bound state are boxed with blue dotted rectangles or ellipses and those involved in the REC–DBD interface are labelled in green. (d) All the residues with two resonance peaks are mapped on the structure (in red), showing that they are mainly located within the DBD–DBD and DBD–DNA interfaces.
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f4: NMR investigations of the PmrA–DNA complex.The overlaid two-dimensional (2D) 1H, 15N TROSY-HSQC spectra (a) and 2D 1H, 13C HSQC spectra, showing the methyl resonances of Ile (b), Leu and Val (c), acquired from PmrA in the absence (black) and presence of DNA (red). The residues that show two sets of amide or methyl resonances in the DNA-bound state are boxed with blue dotted rectangles or ellipses and those involved in the REC–DBD interface are labelled in green. (d) All the residues with two resonance peaks are mapped on the structure (in red), showing that they are mainly located within the DBD–DBD and DBD–DNA interfaces.

Mentions: We successfully prepared the methyl-protonated {Ile(δ1 13CH3), Leu(13CH3, 12CD3), Val(13CH3, 12CD3)} U-[2H, 13C, 15N] sample of PmrA at pH 8.0 and identified 193 of 217 backbone amide resonances (with six Pro residues excluded; Fig. 4a). The 24 missing peaks are mainly located in the flexible regions, such as the N terminus (residues 1–2), the linker region (residues 117–125) and the transactivation loop of DBD (residues 181, 183, 187 and 189–192). The assignment of methyl resonances involved the ‘out-and-back' methyl-detected 3D HMCM(CG)CBCA experiment21, with 13/13 (100%) Ile δ1 methyl resonances, 69/72 (96%) Leu methyl resonances and 24/24 (100%) Val methyl resonances clearly identified (Fig. 4b,c). In BeF3−-activated PmrA, all amide and methyl resonances exhibit only one set of resonance peaks, and the REC and DBD domains do not closely interact with each other because the amide peaks from the stand-alone BeF3−-activated REC dimer and stand-alone DBD domain superimpose well with the peaks from BeF3−-activated PmrA (Supplementary Fig. 9).


Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

Lou YC, Weng TH, Li YC, Kao YF, Lin WF, Peng HL, Chou SH, Hsiao CD, Chen C - Nat Commun (2015)

NMR investigations of the PmrA–DNA complex.The overlaid two-dimensional (2D) 1H, 15N TROSY-HSQC spectra (a) and 2D 1H, 13C HSQC spectra, showing the methyl resonances of Ile (b), Leu and Val (c), acquired from PmrA in the absence (black) and presence of DNA (red). The residues that show two sets of amide or methyl resonances in the DNA-bound state are boxed with blue dotted rectangles or ellipses and those involved in the REC–DBD interface are labelled in green. (d) All the residues with two resonance peaks are mapped on the structure (in red), showing that they are mainly located within the DBD–DBD and DBD–DNA interfaces.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660055&req=5

f4: NMR investigations of the PmrA–DNA complex.The overlaid two-dimensional (2D) 1H, 15N TROSY-HSQC spectra (a) and 2D 1H, 13C HSQC spectra, showing the methyl resonances of Ile (b), Leu and Val (c), acquired from PmrA in the absence (black) and presence of DNA (red). The residues that show two sets of amide or methyl resonances in the DNA-bound state are boxed with blue dotted rectangles or ellipses and those involved in the REC–DBD interface are labelled in green. (d) All the residues with two resonance peaks are mapped on the structure (in red), showing that they are mainly located within the DBD–DBD and DBD–DNA interfaces.
Mentions: We successfully prepared the methyl-protonated {Ile(δ1 13CH3), Leu(13CH3, 12CD3), Val(13CH3, 12CD3)} U-[2H, 13C, 15N] sample of PmrA at pH 8.0 and identified 193 of 217 backbone amide resonances (with six Pro residues excluded; Fig. 4a). The 24 missing peaks are mainly located in the flexible regions, such as the N terminus (residues 1–2), the linker region (residues 117–125) and the transactivation loop of DBD (residues 181, 183, 187 and 189–192). The assignment of methyl resonances involved the ‘out-and-back' methyl-detected 3D HMCM(CG)CBCA experiment21, with 13/13 (100%) Ile δ1 methyl resonances, 69/72 (96%) Leu methyl resonances and 24/24 (100%) Val methyl resonances clearly identified (Fig. 4b,c). In BeF3−-activated PmrA, all amide and methyl resonances exhibit only one set of resonance peaks, and the REC and DBD domains do not closely interact with each other because the amide peaks from the stand-alone BeF3−-activated REC dimer and stand-alone DBD domain superimpose well with the peaks from BeF3−-activated PmrA (Supplementary Fig. 9).

Bottom Line: However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution.Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression.We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.

ABSTRACT
PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

Show MeSH
Related in: MedlinePlus