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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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DBD–DBD interface and conformational changes in DBD.(a) The interactions between two DBDs bound with the promoter DNA. The DBD in PmrA-1 is on the left and in PmrA-2 is on the right. The side chains that form salt bridge interactions are shown with sticks and those that form hydrophobic contacts are yellow sticks and surfaces. (b) Comparison of structures of isolated PmrA DBD (green) and the DBD in PmrA-1 (blue). The r.m.s.d. value between two structures is 1.02 Å for Cα atoms from residues 127–178 (β6 to α7), which suggests that the DNA binding and DBD–DBD interaction altered the conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin.
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f2: DBD–DBD interface and conformational changes in DBD.(a) The interactions between two DBDs bound with the promoter DNA. The DBD in PmrA-1 is on the left and in PmrA-2 is on the right. The side chains that form salt bridge interactions are shown with sticks and those that form hydrophobic contacts are yellow sticks and surfaces. (b) Comparison of structures of isolated PmrA DBD (green) and the DBD in PmrA-1 (blue). The r.m.s.d. value between two structures is 1.02 Å for Cα atoms from residues 127–178 (β6 to α7), which suggests that the DNA binding and DBD–DBD interaction altered the conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin.

Mentions: The two DBDs fit complementarily to each other with an interface of 291.8 Å2 in complex-1. The C-terminal β-hairpin and the loop between α6 and α7 of the PmrA-1 DBD interacts with the β7–β8 loop of the PmrA-2 DBD (Fig. 2a). The side chains of Arg207 (PmrA-1) and Asp149 (PmrA-2) establish a salt bridge, and those of Ser167 (PmrA-1) and Arg138 (PmrA-2) form an H-bond. Also, Pro168 and Phe212 from PmrA-1 as well as Arg139 and Leu140 from PmrA-2 constitute a hydrophobic cluster. Complex-2 and complex-3 feature similar interactions between the two DBDs, with interfaces of 303.7 and 284.1 Å2, respectively. We compared the structures of DBD in the free and DNA-bound states. The r.m.s.d. value between PmrA-1 DBD in complex-1 and the free stand-alone DBD NMR structure is large, with 2.26 Å for Cα atoms from residues 127–216. However, the r.m.s.d. value decreases to 1.02 Å if only residues 127–178 (the N-terminal β-sheet to helix α7) are superimposed (Fig. 2b). The conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin are altered when DBD binds to DNA.


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)

DBD–DBD interface and conformational changes in DBD.(a) The interactions between two DBDs bound with the promoter DNA. The DBD in PmrA-1 is on the left and in PmrA-2 is on the right. The side chains that form salt bridge interactions are shown with sticks and those that form hydrophobic contacts are yellow sticks and surfaces. (b) Comparison of structures of isolated PmrA DBD (green) and the DBD in PmrA-1 (blue). The r.m.s.d. value between two structures is 1.02 Å for Cα atoms from residues 127–178 (β6 to α7), which suggests that the DNA binding and DBD–DBD interaction altered the conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: DBD–DBD interface and conformational changes in DBD.(a) The interactions between two DBDs bound with the promoter DNA. The DBD in PmrA-1 is on the left and in PmrA-2 is on the right. The side chains that form salt bridge interactions are shown with sticks and those that form hydrophobic contacts are yellow sticks and surfaces. (b) Comparison of structures of isolated PmrA DBD (green) and the DBD in PmrA-1 (blue). The r.m.s.d. value between two structures is 1.02 Å for Cα atoms from residues 127–178 (β6 to α7), which suggests that the DNA binding and DBD–DBD interaction altered the conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin.
Mentions: The two DBDs fit complementarily to each other with an interface of 291.8 Å2 in complex-1. The C-terminal β-hairpin and the loop between α6 and α7 of the PmrA-1 DBD interacts with the β7–β8 loop of the PmrA-2 DBD (Fig. 2a). The side chains of Arg207 (PmrA-1) and Asp149 (PmrA-2) establish a salt bridge, and those of Ser167 (PmrA-1) and Arg138 (PmrA-2) form an H-bond. Also, Pro168 and Phe212 from PmrA-1 as well as Arg139 and Leu140 from PmrA-2 constitute a hydrophobic cluster. Complex-2 and complex-3 feature similar interactions between the two DBDs, with interfaces of 303.7 and 284.1 Å2, respectively. We compared the structures of DBD in the free and DNA-bound states. The r.m.s.d. value between PmrA-1 DBD in complex-1 and the free stand-alone DBD NMR structure is large, with 2.26 Å for Cα atoms from residues 127–216. However, the r.m.s.d. value decreases to 1.02 Å if only residues 127–178 (the N-terminal β-sheet to helix α7) are superimposed (Fig. 2b). The conformations of the transactivation loop, the recognition helix α8 and the C-terminal β-hairpin are altered when DBD binds to DNA.

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