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Binding interface between the Salmonella σ(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

Cavaliere P, Sizun C, Levi-Acobas F, Nowakowski M, Monteil V, Bontems F, Bellalou J, Mayer C, Norel F - Sci Rep (2015)

Bottom Line: Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S.The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface.This study provides the basis for further structural investigation of the σ(S)-Crl complex.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Laboratoire Systèmes Macromoléculaires et Signalisation, Département de Microbiologie, 25 rue du Docteur Roux, 75015 Paris, France.

ABSTRACT
In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its association to E. Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S. Typhimurium σ(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ(S) and to generate in silico models of the σ(S)-Crl complex constrained by mutational analysis. The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ(S)-Crl complex.

No MeSH data available.


Related in: MedlinePlus

Structural analysis by NMR of the D36A CrlSTM mutant.(a) Selected region of assigned 1H-15N HSQC spectra of 13C15N-labeled D36A (red) and wild-type (black) CrlSTM displaying chemical shift perturbations due to the D36A substitution. Red lines link D36A to the corresponding wild-type CrlSTM signals. (b) Plot of combined 1H15N amide and 13Cα and 13C’ backbone CSPs as a function of residue numbers in CrlSTM. Secondary structures are indicated by background coloring (red for α-helices and blue for β-strands) and annotated on top. Loops are denoted by the letter L. (c) Mapping of amide CSPs on a cartoon representation of an NMR ensemble structure (10 conformers) of CrlSTM. Nitrogen atoms are shown by red and orange spheres for residues with ΔδHN > 0.1 ppm and >0.05 ppm, respectively. The side chains of R24, D36 and W82 are shown as lines in green, cyan and magenta, respectively. The corresponding side chains of a model built from the X-ray structure of CrlPM (PDB 4Q11) are indicated in sticks with the same colours.
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f6: Structural analysis by NMR of the D36A CrlSTM mutant.(a) Selected region of assigned 1H-15N HSQC spectra of 13C15N-labeled D36A (red) and wild-type (black) CrlSTM displaying chemical shift perturbations due to the D36A substitution. Red lines link D36A to the corresponding wild-type CrlSTM signals. (b) Plot of combined 1H15N amide and 13Cα and 13C’ backbone CSPs as a function of residue numbers in CrlSTM. Secondary structures are indicated by background coloring (red for α-helices and blue for β-strands) and annotated on top. Loops are denoted by the letter L. (c) Mapping of amide CSPs on a cartoon representation of an NMR ensemble structure (10 conformers) of CrlSTM. Nitrogen atoms are shown by red and orange spheres for residues with ΔδHN > 0.1 ppm and >0.05 ppm, respectively. The side chains of R24, D36 and W82 are shown as lines in green, cyan and magenta, respectively. The corresponding side chains of a model built from the X-ray structure of CrlPM (PDB 4Q11) are indicated in sticks with the same colours.

Mentions: The NMR ensemble structure (Fig. 5a) showed that several regions at the periphery of the core display structural disorder. NMR signals in loop 1 (L19-F33) were broad, possibly due to conformational exchange at the millisecond timescale. Still a number of NOE contacts were found with α1 and β2, showing that it is not completely disordered. Due to the absence of the small helix α2 found in CrlPM, loop 1 of CrlSTM explores a wider space and contributes to forming a deeper cavity than in CrlPM (Supplementary Fig. S6). Residues in loop 2 displayed sharp signals but only few NOE contacts, indicating that this region is disordered and flexible. The difference of dynamics in loop 2 as compared to the structured regions was also corroborated by 15N relaxation experiments, where it displays lower R2 rates that deviate from simulated R2 values (Supplementary Fig. S7). Finally the region corresponding to helix α4 in CrlPM is not structured in CrlSTM (Supplementary Fig. S6b). Indeed, only few inter-residue NOE contacts were found in the P120-P128 region, but they provided evidence of the proximity between the C-terminus and helix α3. Strikingly, signals of several residues in the core β-sheet were broad, denoting conformational fluctuations that might be coupled to those in loop 1. The corresponding side chains could not be constrained during structure calculation, prominently that of W82 in strand β4, which points towards the cavity in the crystal structure, but appears to flip out in the NMR structures (Fig. 6c).


Binding interface between the Salmonella σ(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

Cavaliere P, Sizun C, Levi-Acobas F, Nowakowski M, Monteil V, Bontems F, Bellalou J, Mayer C, Norel F - Sci Rep (2015)

Structural analysis by NMR of the D36A CrlSTM mutant.(a) Selected region of assigned 1H-15N HSQC spectra of 13C15N-labeled D36A (red) and wild-type (black) CrlSTM displaying chemical shift perturbations due to the D36A substitution. Red lines link D36A to the corresponding wild-type CrlSTM signals. (b) Plot of combined 1H15N amide and 13Cα and 13C’ backbone CSPs as a function of residue numbers in CrlSTM. Secondary structures are indicated by background coloring (red for α-helices and blue for β-strands) and annotated on top. Loops are denoted by the letter L. (c) Mapping of amide CSPs on a cartoon representation of an NMR ensemble structure (10 conformers) of CrlSTM. Nitrogen atoms are shown by red and orange spheres for residues with ΔδHN > 0.1 ppm and >0.05 ppm, respectively. The side chains of R24, D36 and W82 are shown as lines in green, cyan and magenta, respectively. The corresponding side chains of a model built from the X-ray structure of CrlPM (PDB 4Q11) are indicated in sticks with the same colours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559669&req=5

f6: Structural analysis by NMR of the D36A CrlSTM mutant.(a) Selected region of assigned 1H-15N HSQC spectra of 13C15N-labeled D36A (red) and wild-type (black) CrlSTM displaying chemical shift perturbations due to the D36A substitution. Red lines link D36A to the corresponding wild-type CrlSTM signals. (b) Plot of combined 1H15N amide and 13Cα and 13C’ backbone CSPs as a function of residue numbers in CrlSTM. Secondary structures are indicated by background coloring (red for α-helices and blue for β-strands) and annotated on top. Loops are denoted by the letter L. (c) Mapping of amide CSPs on a cartoon representation of an NMR ensemble structure (10 conformers) of CrlSTM. Nitrogen atoms are shown by red and orange spheres for residues with ΔδHN > 0.1 ppm and >0.05 ppm, respectively. The side chains of R24, D36 and W82 are shown as lines in green, cyan and magenta, respectively. The corresponding side chains of a model built from the X-ray structure of CrlPM (PDB 4Q11) are indicated in sticks with the same colours.
Mentions: The NMR ensemble structure (Fig. 5a) showed that several regions at the periphery of the core display structural disorder. NMR signals in loop 1 (L19-F33) were broad, possibly due to conformational exchange at the millisecond timescale. Still a number of NOE contacts were found with α1 and β2, showing that it is not completely disordered. Due to the absence of the small helix α2 found in CrlPM, loop 1 of CrlSTM explores a wider space and contributes to forming a deeper cavity than in CrlPM (Supplementary Fig. S6). Residues in loop 2 displayed sharp signals but only few NOE contacts, indicating that this region is disordered and flexible. The difference of dynamics in loop 2 as compared to the structured regions was also corroborated by 15N relaxation experiments, where it displays lower R2 rates that deviate from simulated R2 values (Supplementary Fig. S7). Finally the region corresponding to helix α4 in CrlPM is not structured in CrlSTM (Supplementary Fig. S6b). Indeed, only few inter-residue NOE contacts were found in the P120-P128 region, but they provided evidence of the proximity between the C-terminus and helix α3. Strikingly, signals of several residues in the core β-sheet were broad, denoting conformational fluctuations that might be coupled to those in loop 1. The corresponding side chains could not be constrained during structure calculation, prominently that of W82 in strand β4, which points towards the cavity in the crystal structure, but appears to flip out in the NMR structures (Fig. 6c).

Bottom Line: Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S.The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface.This study provides the basis for further structural investigation of the σ(S)-Crl complex.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Laboratoire Systèmes Macromoléculaires et Signalisation, Département de Microbiologie, 25 rue du Docteur Roux, 75015 Paris, France.

ABSTRACT
In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its association to E. Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S. Typhimurium σ(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ(S) and to generate in silico models of the σ(S)-Crl complex constrained by mutational analysis. The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ(S)-Crl complex.

No MeSH data available.


Related in: MedlinePlus