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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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Weighted chemical shift perturbations (CSPs) and NMR dynamics on PmrA with and without DNA.CSP values for backbone amide resonances (a) and for methyl resonances (b). The black bars represent the residues that can be identified in the DNA-bound state but are missing without DNA. The dotted lines indicate the mean Δδ value plus 1 s.d. of Δδ values. (c) The R1 and R2 values of BeF3−-activated PmrA are measured in duplicate and the R2/R1 values are calculated. The average R2/R1 values are 72.5 and 40.1 for residues in REC and DBD, respectively, and are marked by dotted lines. Error bars represent fitting errors. (d) The intensity ratios of amide resonances in the DNA-bound state to free state. Error bars are noise-to-signal ratios. The mean intensity ratios are 0.46±0.10 for REC and 0.19±0.09 for DBD as indicated by the dotted lines.
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f5: Weighted chemical shift perturbations (CSPs) and NMR dynamics on PmrA with and without DNA.CSP values for backbone amide resonances (a) and for methyl resonances (b). The black bars represent the residues that can be identified in the DNA-bound state but are missing without DNA. The dotted lines indicate the mean Δδ value plus 1 s.d. of Δδ values. (c) The R1 and R2 values of BeF3−-activated PmrA are measured in duplicate and the R2/R1 values are calculated. The average R2/R1 values are 72.5 and 40.1 for residues in REC and DBD, respectively, and are marked by dotted lines. Error bars represent fitting errors. (d) The intensity ratios of amide resonances in the DNA-bound state to free state. Error bars are noise-to-signal ratios. The mean intensity ratios are 0.46±0.10 for REC and 0.19±0.09 for DBD as indicated by the dotted lines.

Mentions: In the DNA-bound state, many amides and methyl groups in DBD show two sets of resonance peaks, including Asn188, Thr189, Glu191, Ile194, Arg210 and Gly211, which bind to different DNA sequences in the crystal structure, and Thr137, Leu140, Trp142, Gly144, Asp149, Gly166, Arg207 and Thr208, which are located in the DBD–DBD interface (Fig. 4a–c). We mapped all of the residues with two resonance peaks on the structure, showing that these residues are mainly located within the DBD–DBD and DBD–DNA interfaces (Fig. 4d). Calculation of the weighted chemical shift perturbations (CSPs) of the amide and methyl resonances with and without DNA (Fig. 5a,b) shows that the residues with significant CSPs (Δδ>Δδaverage+s.d.) are also mostly located within the DBD–DBD and DBD–DNA interfaces. In the PmrA–DNA complex structure, the two DBDs bind to the half-1 and half-2 sites to constitute a head-to-tail interface. Therefore, the asymmetric DBD–DBD and DBD–DNA interactions between two PmrA protomers in complex with DNA are also observed on NMR.


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)

Weighted chemical shift perturbations (CSPs) and NMR dynamics on PmrA with and without DNA.CSP values for backbone amide resonances (a) and for methyl resonances (b). The black bars represent the residues that can be identified in the DNA-bound state but are missing without DNA. The dotted lines indicate the mean Δδ value plus 1 s.d. of Δδ values. (c) The R1 and R2 values of BeF3−-activated PmrA are measured in duplicate and the R2/R1 values are calculated. The average R2/R1 values are 72.5 and 40.1 for residues in REC and DBD, respectively, and are marked by dotted lines. Error bars represent fitting errors. (d) The intensity ratios of amide resonances in the DNA-bound state to free state. Error bars are noise-to-signal ratios. The mean intensity ratios are 0.46±0.10 for REC and 0.19±0.09 for DBD as indicated by the dotted lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Weighted chemical shift perturbations (CSPs) and NMR dynamics on PmrA with and without DNA.CSP values for backbone amide resonances (a) and for methyl resonances (b). The black bars represent the residues that can be identified in the DNA-bound state but are missing without DNA. The dotted lines indicate the mean Δδ value plus 1 s.d. of Δδ values. (c) The R1 and R2 values of BeF3−-activated PmrA are measured in duplicate and the R2/R1 values are calculated. The average R2/R1 values are 72.5 and 40.1 for residues in REC and DBD, respectively, and are marked by dotted lines. Error bars represent fitting errors. (d) The intensity ratios of amide resonances in the DNA-bound state to free state. Error bars are noise-to-signal ratios. The mean intensity ratios are 0.46±0.10 for REC and 0.19±0.09 for DBD as indicated by the dotted lines.
Mentions: In the DNA-bound state, many amides and methyl groups in DBD show two sets of resonance peaks, including Asn188, Thr189, Glu191, Ile194, Arg210 and Gly211, which bind to different DNA sequences in the crystal structure, and Thr137, Leu140, Trp142, Gly144, Asp149, Gly166, Arg207 and Thr208, which are located in the DBD–DBD interface (Fig. 4a–c). We mapped all of the residues with two resonance peaks on the structure, showing that these residues are mainly located within the DBD–DBD and DBD–DNA interfaces (Fig. 4d). Calculation of the weighted chemical shift perturbations (CSPs) of the amide and methyl resonances with and without DNA (Fig. 5a,b) shows that the residues with significant CSPs (Δδ>Δδaverage+s.d.) are also mostly located within the DBD–DBD and DBD–DNA interfaces. In the PmrA–DNA complex structure, the two DBDs bind to the half-1 and half-2 sites to constitute a head-to-tail interface. Therefore, the asymmetric DBD–DBD and DBD–DNA interactions between two PmrA protomers in complex with DNA are also observed on NMR.

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