Mol. Cells 2017; 40(4): 299-306
Published online April 20, 2017
https://doi.org/10.14348/molcells.2017.0015
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: choish@snu.ac.kr (SHC); hanc210@snu.ac.kr (NCH)
The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens
Keywords crystal structure, low pH, transcriptional regulator AphB,
Many pathogenic bacteria increase the expression of virulence factors by recognizing and responding to the host environment.
LTTRs comprise one of the largest families of transcriptional regulators in prokaryotes that are involved in diverse biological processes. They are composed of an N-terminal DNA binding domain (DBD) and a C-terminal regulatory domain (RD). Crystal structures of full-length AphB from
In
AphB of
DNA encoding the AphB RD (residues 88-291) was amplified by PCR using the
The cysteine codon at position 227 (C227) was changed to a serine codon (C227S) using the overlapping PCR method with Pfu polymerase based on pProEx-HTa-VvAphB-RD (Patel et al., 1993).
Crystallization of the wild type AphB RD and RD treated with the oxidant cumene hydroperoxide (CHP) was performed using the vapor-diffusion hanging drop method at 14°C under a mother liquor containing 0.1 M HEPES (pH 7.5), 15% (
Crystallization of the AphB C227S mutant RD was performed using the same method as described above with a mother liquor containing 0.35 M potassium thiocyanate (pH 7.0) and 17% (
pJR0325, which was constructed previously to carry a mutant allele of
The wild type and
The wild type and the
We initially attempted to obtain the full-length AphB from
Like other LTTR RD structures, the
Importantly, a small cavity was found at the interface between RD-I and RD-II, which was also observed in the
We noted the local chemical environment around C227 in RD-II, which is the only cysteine residue in the
To assess the structural impact of the C227 sulfur atom in
In order to determine reactivity of the cysteine residue, we incubated the crystals with various peroxides such as hydrogen peroxide and alkyl hydroperoxides and determined the structures. No oxidation modification at the cysteine residue was found in the structures under any of the tested conditions (Fig. 3C). In the structure of
Both
We next carried out qRT-PCR to examine pH-dependent AphB transcriptional activity. When we measured
In this study, we determined the crystal structures of
Since AphB senses anoxic environments, the cysteine residue (C227) was investigated based on the C227S mutant structure and the reaction by various peroxide molecules. The mutation of C227S did not give substantial structural alteration to AphB. Furthermore, incubation of peroxides did not result in any oxidation on the cysteine residue. Instead, the CHP-treated structure showed a new cavity on the opposite side of the RD dimer with an unidentified ligand molecule in it. Taken together, our findings suggest that the cysteine in AphB RD might not directly participate in sensing oxygen.
AphB was responsive to low pH as well as low oxygen tension in
Then, how are the anoxic conditions recognized by AphB in
In this study, we presented the crystal structures of AphB RD of
(A) The structure of the wild type
(A)
Superposition of three dimers from the
(A) The front and back sides of the surface representations of the
(A)
Statistics for data collection and refinement
Native | CHP-incubated | C227S | |
---|---|---|---|
Data collection | |||
Beam line | PAL 5C | PAL 5C | PAL 5C |
Wavelength | 0.97960 | 0.97960 | 0.97930 |
Space group | |||
Cell dimensions | |||
| 125.0, 188.0, 57.4 | 124.8, 189.4, 57.4 | 230.3,72.4, 112.2 |
α,β,γ (°) | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 |
Resolution (Å) | 50.0–1.90 (1.93–1.90) | 50.0–2.40 (2.44–2.40) | 50.0–3.0 (3.05–3.00) |
Rmerge | 0.111 (0.639) | 0.122 (0.433) | 0.169 (0.445) |
| 14.1 (3.7) | 13.4 (3.0) | 7.2 (2.1) |
Completeness (%) | 98.1 (85.4) | 99.7 (99.4) | 97.2 (90.6) |
Redundancy | 8.7 (6.2) | 8.8 (5.7) | 4.2 (2.6) |
Refinement | |||
Resolution (Å) | 42.26–1.90 | 45.70–2.40 | 38.93 – 3.00 |
No. reflections | 90980 | 51444 | 28983 |
Rwork/Rfree | 0.219/0.263 | 0.21/0.26 | 0.24/0.29 |
No. of total atoms | 9655 | 9382 | 9186 |
Wilson B-factor (Å) | 19.40 | 30.79 | 47.55 |
R.M.S deviations | |||
Bond lengths (Å) | 0.005 | 0.003 | 0.003 |
Bond angles (°) | 1.11 | 0.56 | 0.60 |
Ramachandran plot | |||
Favored (%) | 98.3 | 97.1 | 94.9 |
Allowed (%) | 1.8 | 2.9 | 5.1 |
Outliers (%) | 0.00 | 0.00 | 0.00 |
PDB ID | 5FHK | 5X0O | 5X0N |
Values in parentheses are for the highest resolution shell.
**R merge = ∑ hkl ∑ i |I i (hkl) – [I(hkl)]|/∑ hkl ∑ i I i (hkl), where I i (hkl) is the intensity of the ith observation of reflection hkl and [I(hkl)] is the average intensity of the i observations.
***R free calculated for a random set of 10% of reflections not used in the refinement
Plasmids and bacterial strains used in this study
Strain or plasmid | Relevant characteristics a | Reference or source |
---|---|---|
Bacterial strains | ||
| ||
MO6-24/O | Clinical isolate; virulent | Wright et al., 1990 |
KK1419 | MO6-24/O with Δ | This study |
| ||
S17-1λ | λ- | Simon et al., 1983 |
C43 (DE3) | F− | Miroux and Walker, 1996 |
Plasmids | ||
pProEx-HTa-VvAphB-RD | His6–tag fusion protein expression vector; Apr | Invitrogen |
pDM4 | R6K γ | Milton et al., 1996 |
pJR0325 | pDM4 with Δ | Rhee et al., 2006 |
aTpr, trimethoprim resistance; Smr, streptomycin resistance; Apr, ampicillin resistance; Cmr, chloramphenicol resistance
Mol. Cells 2017; 40(4): 299-306
Published online April 30, 2017 https://doi.org/10.14348/molcells.2017.0015
Copyright © The Korean Society for Molecular and Cellular Biology.
Nohra Park1,3, Saemee Song1,3, Garam Choi1,2, Kyung Ku Jang1,2, Inseong Jo1, Sang Ho Choi1,2,*, and Nam-Chul Ha1,*
1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea, 2National Research Laboratory of Molecular Microbiology and Toxicology, Seoul National University, Seoul 08826, Korea
Correspondence to:*Correspondence: choish@snu.ac.kr (SHC); hanc210@snu.ac.kr (NCH)
The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens
Keywords: crystal structure, low pH, transcriptional regulator AphB,
Many pathogenic bacteria increase the expression of virulence factors by recognizing and responding to the host environment.
LTTRs comprise one of the largest families of transcriptional regulators in prokaryotes that are involved in diverse biological processes. They are composed of an N-terminal DNA binding domain (DBD) and a C-terminal regulatory domain (RD). Crystal structures of full-length AphB from
In
AphB of
DNA encoding the AphB RD (residues 88-291) was amplified by PCR using the
The cysteine codon at position 227 (C227) was changed to a serine codon (C227S) using the overlapping PCR method with Pfu polymerase based on pProEx-HTa-VvAphB-RD (Patel et al., 1993).
Crystallization of the wild type AphB RD and RD treated with the oxidant cumene hydroperoxide (CHP) was performed using the vapor-diffusion hanging drop method at 14°C under a mother liquor containing 0.1 M HEPES (pH 7.5), 15% (
Crystallization of the AphB C227S mutant RD was performed using the same method as described above with a mother liquor containing 0.35 M potassium thiocyanate (pH 7.0) and 17% (
pJR0325, which was constructed previously to carry a mutant allele of
The wild type and
The wild type and the
We initially attempted to obtain the full-length AphB from
Like other LTTR RD structures, the
Importantly, a small cavity was found at the interface between RD-I and RD-II, which was also observed in the
We noted the local chemical environment around C227 in RD-II, which is the only cysteine residue in the
To assess the structural impact of the C227 sulfur atom in
In order to determine reactivity of the cysteine residue, we incubated the crystals with various peroxides such as hydrogen peroxide and alkyl hydroperoxides and determined the structures. No oxidation modification at the cysteine residue was found in the structures under any of the tested conditions (Fig. 3C). In the structure of
Both
We next carried out qRT-PCR to examine pH-dependent AphB transcriptional activity. When we measured
In this study, we determined the crystal structures of
Since AphB senses anoxic environments, the cysteine residue (C227) was investigated based on the C227S mutant structure and the reaction by various peroxide molecules. The mutation of C227S did not give substantial structural alteration to AphB. Furthermore, incubation of peroxides did not result in any oxidation on the cysteine residue. Instead, the CHP-treated structure showed a new cavity on the opposite side of the RD dimer with an unidentified ligand molecule in it. Taken together, our findings suggest that the cysteine in AphB RD might not directly participate in sensing oxygen.
AphB was responsive to low pH as well as low oxygen tension in
Then, how are the anoxic conditions recognized by AphB in
In this study, we presented the crystal structures of AphB RD of
(A) The structure of the wild type
(A)
Superposition of three dimers from the
(A) The front and back sides of the surface representations of the
(A)
. Statistics for data collection and refinement.
Native | CHP-incubated | C227S | |
---|---|---|---|
Data collection | |||
Beam line | PAL 5C | PAL 5C | PAL 5C |
Wavelength | 0.97960 | 0.97960 | 0.97930 |
Space group | |||
Cell dimensions | |||
| 125.0, 188.0, 57.4 | 124.8, 189.4, 57.4 | 230.3,72.4, 112.2 |
α,β,γ (°) | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 |
Resolution (Å) | 50.0–1.90 (1.93–1.90) | 50.0–2.40 (2.44–2.40) | 50.0–3.0 (3.05–3.00) |
Rmerge | 0.111 (0.639) | 0.122 (0.433) | 0.169 (0.445) |
| 14.1 (3.7) | 13.4 (3.0) | 7.2 (2.1) |
Completeness (%) | 98.1 (85.4) | 99.7 (99.4) | 97.2 (90.6) |
Redundancy | 8.7 (6.2) | 8.8 (5.7) | 4.2 (2.6) |
Refinement | |||
Resolution (Å) | 42.26–1.90 | 45.70–2.40 | 38.93 – 3.00 |
No. reflections | 90980 | 51444 | 28983 |
Rwork/Rfree | 0.219/0.263 | 0.21/0.26 | 0.24/0.29 |
No. of total atoms | 9655 | 9382 | 9186 |
Wilson B-factor (Å) | 19.40 | 30.79 | 47.55 |
R.M.S deviations | |||
Bond lengths (Å) | 0.005 | 0.003 | 0.003 |
Bond angles (°) | 1.11 | 0.56 | 0.60 |
Ramachandran plot | |||
Favored (%) | 98.3 | 97.1 | 94.9 |
Allowed (%) | 1.8 | 2.9 | 5.1 |
Outliers (%) | 0.00 | 0.00 | 0.00 |
PDB ID | 5FHK | 5X0O | 5X0N |
Values in parentheses are for the highest resolution shell..
**R merge = ∑ hkl ∑ i |I i (hkl) – [I(hkl)]|/∑ hkl ∑ i I i (hkl), where I i (hkl) is the intensity of the ith observation of reflection hkl and [I(hkl)] is the average intensity of the i observations.
***R free calculated for a random set of 10% of reflections not used in the refinement
. Plasmids and bacterial strains used in this study.
Strain or plasmid | Relevant characteristics a | Reference or source |
---|---|---|
Bacterial strains | ||
| ||
MO6-24/O | Clinical isolate; virulent | Wright et al., 1990 |
KK1419 | MO6-24/O with Δ | This study |
| ||
S17-1λ | λ- | Simon et al., 1983 |
C43 (DE3) | F− | Miroux and Walker, 1996 |
Plasmids | ||
pProEx-HTa-VvAphB-RD | His6–tag fusion protein expression vector; Apr | Invitrogen |
pDM4 | R6K γ | Milton et al., 1996 |
pJR0325 | pDM4 with Δ | Rhee et al., 2006 |
aTpr, trimethoprim resistance; Smr, streptomycin resistance; Apr, ampicillin resistance; Cmr, chloramphenicol resistance
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(A) The structure of the wild type
(A)
Superposition of three dimers from the
(A) The front and back sides of the surface representations of the
(A)