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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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REC–DBD interdomain dynamics of PmrA and its possible role in RNAPH interaction.(a) The docking model of PmrA–DNA–RNAPH complex with the REC of PmrA-1 is in magenta, PmrA-2 in orange and DNA in yellow. Surface charge distributions of two DBDs show the acidic patches. The basic patches on σ4 and β-flap tip helix of RNAPH (green) are shown in sticks. (b) Top view of the model with two α-subunits of RNAPH in pink and cyan and others in grey, showing that with stable REC–DBD interface, only the REC on PmrA-1 can interact with the RNAPH. However, if the REC dimer is connected to DBD with flexible linkers, the dimer can tumble freely and find the best orientation to interact extensively with RNAPH. In addition to these interactions, the C-terminal domain of the α-subunit of RNAPH may interact with the upstream DNA or with the PmrA–DNA complex. (c) A cartoon diagram summarizes the structure and dynamics of PmrA in transcription regulation. The REC is in blue and DBD in green. The phosphoryl analogue BeF3− is denoted as P. Linkers with high flexibility are shown as dotted lines and low mobility as solid lines. The interactions between domains are shown as red lines and the domains that can rotate independently by yellow curved arrows.
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f8: REC–DBD interdomain dynamics of PmrA and its possible role in RNAPH interaction.(a) The docking model of PmrA–DNA–RNAPH complex with the REC of PmrA-1 is in magenta, PmrA-2 in orange and DNA in yellow. Surface charge distributions of two DBDs show the acidic patches. The basic patches on σ4 and β-flap tip helix of RNAPH (green) are shown in sticks. (b) Top view of the model with two α-subunits of RNAPH in pink and cyan and others in grey, showing that with stable REC–DBD interface, only the REC on PmrA-1 can interact with the RNAPH. However, if the REC dimer is connected to DBD with flexible linkers, the dimer can tumble freely and find the best orientation to interact extensively with RNAPH. In addition to these interactions, the C-terminal domain of the α-subunit of RNAPH may interact with the upstream DNA or with the PmrA–DNA complex. (c) A cartoon diagram summarizes the structure and dynamics of PmrA in transcription regulation. The REC is in blue and DBD in green. The phosphoryl analogue BeF3− is denoted as P. Linkers with high flexibility are shown as dotted lines and low mobility as solid lines. The interactions between domains are shown as red lines and the domains that can rotate independently by yellow curved arrows.

Mentions: To better understand the roles of the REC–DBD interface in transcription activation, we dock the E. coli RNAPH18 to the PmrA–DNA complex structure on the basis of the crystal structure of the σ4-β-flap tip helix chimer/PhoB–DBD/DNA ternary complex29 (Supplementary Fig. 14). In the PhoB–DBD ternary complex structure, two PhoB–DBDs recognized the two half sites in the head-to-tail orientation and the σ4-β-flap tip helix chimer contacted the −35 element as well as the PhoB–DBD at the half-1 site. In the PmrA–DNA complex structure, two PmrA DBDs bind to promoter DNA sequences in a head-to-tail manner and the −35 element also locates at the half-1 site of the pbgP promoter30. With the similarity in promoter recognition between PmrA and PhoB, the PhoB–DBD ternary complex is a suitable bridge for the docking of RNAPH, although we have no direct evidence that PmrA interacts with σ4 RNAPH as does PhoB. In the docking model of the PmrA–DNA–RNAPH complex, the σ4 from RNAPH fits complementarily to the interface formed by the two PmrA DBDs. The acidic patches (Glu172, Asp177, Asp182 and Glu184) on the transactivation loop of two DBDs face the patch of basic residues from the σ4 and the β-flap tip helix (Fig. 8a). However, in another view of the docking model (Fig. 8b), with extensive REC–DBD interactions in PmrA-1, only the REC of PmrA-1 contacts with the RNAPH. Instead, with flexible linkers, the REC dimer can search for the best orientation to interact with the RNAPH with a larger interface when the DBDs are bound with the promoter DNA (Supplementary Movie 1). The REC–DBD interface seems not to play an important role in the contact between BeF3−-activated PmrA and the RNAPH, which agrees with the β-galactosidase reporter assay findings of PmrA variants with altered interface residues and suggests that the formation of a stable REC–DBD interface is not crucial for activating downstream gene transcription. This model suggests a direction for future investigation in that the REC–DBD interdomain dynamics and the DBD–DBD interface of PmrA may help in the formation of the initial closed promoter complex for transcription initiation.


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)

REC–DBD interdomain dynamics of PmrA and its possible role in RNAPH interaction.(a) The docking model of PmrA–DNA–RNAPH complex with the REC of PmrA-1 is in magenta, PmrA-2 in orange and DNA in yellow. Surface charge distributions of two DBDs show the acidic patches. The basic patches on σ4 and β-flap tip helix of RNAPH (green) are shown in sticks. (b) Top view of the model with two α-subunits of RNAPH in pink and cyan and others in grey, showing that with stable REC–DBD interface, only the REC on PmrA-1 can interact with the RNAPH. However, if the REC dimer is connected to DBD with flexible linkers, the dimer can tumble freely and find the best orientation to interact extensively with RNAPH. In addition to these interactions, the C-terminal domain of the α-subunit of RNAPH may interact with the upstream DNA or with the PmrA–DNA complex. (c) A cartoon diagram summarizes the structure and dynamics of PmrA in transcription regulation. The REC is in blue and DBD in green. The phosphoryl analogue BeF3− is denoted as P. Linkers with high flexibility are shown as dotted lines and low mobility as solid lines. The interactions between domains are shown as red lines and the domains that can rotate independently by yellow curved arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: REC–DBD interdomain dynamics of PmrA and its possible role in RNAPH interaction.(a) The docking model of PmrA–DNA–RNAPH complex with the REC of PmrA-1 is in magenta, PmrA-2 in orange and DNA in yellow. Surface charge distributions of two DBDs show the acidic patches. The basic patches on σ4 and β-flap tip helix of RNAPH (green) are shown in sticks. (b) Top view of the model with two α-subunits of RNAPH in pink and cyan and others in grey, showing that with stable REC–DBD interface, only the REC on PmrA-1 can interact with the RNAPH. However, if the REC dimer is connected to DBD with flexible linkers, the dimer can tumble freely and find the best orientation to interact extensively with RNAPH. In addition to these interactions, the C-terminal domain of the α-subunit of RNAPH may interact with the upstream DNA or with the PmrA–DNA complex. (c) A cartoon diagram summarizes the structure and dynamics of PmrA in transcription regulation. The REC is in blue and DBD in green. The phosphoryl analogue BeF3− is denoted as P. Linkers with high flexibility are shown as dotted lines and low mobility as solid lines. The interactions between domains are shown as red lines and the domains that can rotate independently by yellow curved arrows.
Mentions: To better understand the roles of the REC–DBD interface in transcription activation, we dock the E. coli RNAPH18 to the PmrA–DNA complex structure on the basis of the crystal structure of the σ4-β-flap tip helix chimer/PhoB–DBD/DNA ternary complex29 (Supplementary Fig. 14). In the PhoB–DBD ternary complex structure, two PhoB–DBDs recognized the two half sites in the head-to-tail orientation and the σ4-β-flap tip helix chimer contacted the −35 element as well as the PhoB–DBD at the half-1 site. In the PmrA–DNA complex structure, two PmrA DBDs bind to promoter DNA sequences in a head-to-tail manner and the −35 element also locates at the half-1 site of the pbgP promoter30. With the similarity in promoter recognition between PmrA and PhoB, the PhoB–DBD ternary complex is a suitable bridge for the docking of RNAPH, although we have no direct evidence that PmrA interacts with σ4 RNAPH as does PhoB. In the docking model of the PmrA–DNA–RNAPH complex, the σ4 from RNAPH fits complementarily to the interface formed by the two PmrA DBDs. The acidic patches (Glu172, Asp177, Asp182 and Glu184) on the transactivation loop of two DBDs face the patch of basic residues from the σ4 and the β-flap tip helix (Fig. 8a). However, in another view of the docking model (Fig. 8b), with extensive REC–DBD interactions in PmrA-1, only the REC of PmrA-1 contacts with the RNAPH. Instead, with flexible linkers, the REC dimer can search for the best orientation to interact with the RNAPH with a larger interface when the DBDs are bound with the promoter DNA (Supplementary Movie 1). The REC–DBD interface seems not to play an important role in the contact between BeF3−-activated PmrA and the RNAPH, which agrees with the β-galactosidase reporter assay findings of PmrA variants with altered interface residues and suggests that the formation of a stable REC–DBD interface is not crucial for activating downstream gene transcription. This model suggests a direction for future investigation in that the REC–DBD interdomain dynamics and the DBD–DBD interface of PmrA may help in the formation of the initial closed promoter complex for transcription initiation.

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