Mol. Cells 2019; 42(12): 850-857
Published online November 14, 2019
https://doi.org/10.14348/molcells.2019.0168
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hanc210@snu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The Gram-negative opportunistic pathogen,
Keywords antibiotic resistance, crystal structure, lysR-type transcriptional regulator, MexT, Pseudomonas aeruginosa
The opportunistic human pathogen
The expression level of MexEF-OprN in wild-type strains under laboratory conditions is typically negligible. However, treatment with the fluoroquinolone antibiotic norfloxacin leads to generate certain types of spontaneous mutant strains, such as
We conducted the experiments to evaluate the molecular mechanism of MexT by understanding its structure. We investigated the mechanism by which MexT regulates the expression of MexEF-OprN by producing the full-length (FL) recombinant protein in
The native MexT RD (residues 95–304) and FL MexT (residues 1–304) were expressed as described previously (Kim et al., 2018). For the selenomethionyl (SeMet)-labeled MexT RD, the host
After TALON affinity chromatography, FL MexT was directly loaded onto a size-exclusion chromatography column (HiLoad Superdex 16/60 200; GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 10% (v/v) glycerol, and 2 mM 2-mercaptoethanol. The purified proteins were aliquoted and stored frozen at −173°C.
MexT RD was crystallized using the hanging-drop vapor-diffusion method at 14°C as described previously (Kim et al., 2018). The precipitation solution comprised 0.5 M ammonium sulfate, 1.0 M sodium citrate tribasic dehydrate (pH 5.6), 0.1 M lithium sulfate, and 2 mM ethylenediaminetetraacetic acid (EDTA). Glycerol (30% [v/v], cryoprotectant) was added to the precipitation solution. The X-ray diffraction data were collected in a flash-cooled liquid nitrogen stream at −173°C. SeMet-MexT RD was crystallized under the same crystallization conditions as for native MexT RD. The single-wavelength anomalous diffraction (SAD) dataset was collected from beamline 5C at the Pohang Accelerator Laboratory (Korea) and processed using HKL2000 software (Otwinowski and Minor, 1997).
The 2.0 Å resolution dataset of the native crystal belongs to space group
The sizes of FL MexT and MexT RD were assayed using SEC-MALS. A high-performance liquid chromatography pump (Agilent, USA) was connected to a Superdex-200 10/300 GL gel filtration column (GE Healthcare) and a MALS instrument (Wyatt Dawn Heleos, USA). The size-exclusion chromatography column was pre-equilibrated with buffer comprising 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 2 mM 2-mercaptoethanol for FL MexT; and 20 mM Tris-HCl (pH 8.5), 300 mM NaCl, and 2 mM 2-mercaptoethanol for MexT RD. Bovine serum albumin (2 mg/ml) was used as the standard. MexT RD and FL MexT samples (2 mg/ml) were injected onto the column and eluted at a flow rate of 0.2 ml/min. The datasets were evaluated using the Debye model for fitting static light-scattering data, and refractive index peaks were presented in EASI graphs created using Astra V software (Wyatt Dawn Heleos).
A DNA fragment of 630 bp (the
The PCR product was purified from agarose gel electrophoresis. We mixed 400 ng of DNA and 200 ng of purified FL MexT for 30 min at room temperature. Next, 0.04 units of DNase I (Fermentas) was added, and the reaction mixture (10 mM Tris [pH 8.0], 50 mM KCl, 8 mM MgCl2, 50 ng/μl BSA, 5% glycerol) was incubated for 2 min at room temperature. The reactions were terminated by adding an equal volume of stopping buffer (200 mM NaCl, 1% sodium dodecyl sulfate, 30 mM EDTA), and DNase I was inactivated by heating at 75°C for 10 min. The DNA fragments were recovered via phenol extraction and alcohol precipitation and fragment-sequences were analyzed using an ABI3730xl DNA Analyzer (Applied Biosystems, USA). The size distribution was scanned using Peak Scanner software (ver. 1.0; Applied Biosystems).
The FL MexT (residues 1–304) and its RD (residues 95–304) were produced in
To obtain phase information, we acquired the diffraction dataset using the crystal of the SeMet-labeled protein, which was crystallized in the presence of a high concentration of ammonium sulfate. The structure was determined via the SAD method using the anomalous signals from SeMet, which was refined to the 2.0-Å-resolution native dataset with an
MexT RD protomers are composed of 10 β-strands and 7 α-helices, as in the typical LTTR RD (Jo et al., 2015). Each protomer can be divided into two subdomains—RD-I (residues 95–172, 274–304) and RD-II (residues 173–273). The folding pattern of the two subdomains was similar to Rossman-fold topology; i.e., a central β-sheet bound by helices and loops. The RD-I subdomain comprised three α helices (α1, α2, and α7) and five β-strands (β1, β2, β3, β4, and β10), whereas the RD-II subdomain comprised four α helices (α3, α4, α5, and α6) and five β-strands (β5, β6, β7, β8, and β9). The RD-I and RD-II subdomains were connected by two loops—one connects β4 of RD-I to β5 of RD-II, and the other connects β9 of RD-II to β10 of RD-I (Fig. 2).
The four protomers (chains A–D) in the asymmetric unit are arranged as two of dimers (chains A:B and C:D; Fig. 2). The overall arrangement of the MexT RD dimer was similar to those of other LTTRs. Within crisscrossing protomer dimer, RD-I of one protomer directly interacted with RD-II of the other protomer. The major interactions of two protomers were intermolecular hydrogen bonds between residues in the RD-1 β2 backbone and the RD-II β7 backbone at the dimeric interface. Hydrophobic interactions were found at the dimeric interface of RD-1 α1 and RD-II α5, and likely stabilize the dimer (Supplementary Fig. S1).
The asymmetric unit contained five sulfate ions, which may originate from the crystallization solution (Fig. 3 and Supplementary Fig. S2). Of the five sulfate ions, three were found at the dimeric interfaces; the sulfate ions interacted with Arg134 and Arg135 near the molecular pseudo-2-fold axis—one in the A:B chain dimer and two in the C:D chain dimer (Fig. 3). The fourth sulfate ion is found in the putative ligand-binding site (Fig. 4A). The fifth sulfate ion is located outside of the A:B chain dimer, interacting with Arg120 and Arg121 of chain A (Supplementary Fig. S2).
The dimeric arrangements of the two dimers were similar (Supplementary Fig. S3), but the relative position of subdomain RD-II within the overall frame showed ~5.0 Å deviation. The orientation of the protomers in the dimer can be used to distinguish active from inactive LTTRs (Choi et al., 2001; Jo et al., 2015). To examine the conformation of the RD dimer, the obtained MexT RD dimer (chains A:B) was superimposed onto the
Compared to a typical LTTR
As mentioned above, one sulfate ion is located at the putative ligand-binding site (Fig. 3A). Arg220 and Arg269 ionically interact with the sulfate ion at the putative ligand-binding site of chain B (Fig. 3A). In particular, guanidine group of Arg269 in chain B can make two configurations by flipping. And it is the site where one sulfate ion can bind. Pocket-formation by the flipping guanidine group makes space for holding small molecules. The sulfate-binding region extends to the hydrophobic surface region decorated by Tyr138, Phe201, Phe208, and Phe229 (Fig. 4B and Supplementary Fig. S5). Highlighting the space of adjacent sulfate-binding region and hydrophobic patches in the ligand-binding site, our structure suggests that candidate ligands should have a negatively charged (or polar) moiety linked to a hydrophobic moiety.
Cinnamaldehyde upregulated the expression of MexEF-OprN. Indeed, cinnamaldehyde has a polar aldehyde moiety and a hydrophobic phenyl ring (Juarez et al., 2017). The polar aldehyde moiety likely interacts with the positively charged pocket, which is composed of Arg220 and Arg269, and the hydrophobic phenyl ring probably participates in the interaction. A molecular model showed that cinnamaldehyde fitted the pocket well (Dallakyan and Olson, 2015) (Supplementary Fig. S6). Molecules with such characteristics may activate MexT and alter its association with DNA.
To elucidate the interaction between MexT with the promoter of
Next, the two MexT-binding site sequences identified by DNase I footprinting were aligned with the promoter sequences of other genes in the MexT regulon (PA1744, PA1970, PA2486, and PA2759). The MexT regulon was identified with microarray-based transcriptome profiling (Tian et al., 2009), and was further experimentally confirmed by electromobility shift assays and β-galactosidase assays using transcriptional fusion reporters (Maseda et al., 2010; Tian et al., 2009). The sequence alignment showed a palindromic consensus pattern of ATCA(N7)CGAT at each MexT-binding site. Notably, there was partial overlap between the front region
The MexT-binding sequence has been predicted in previous studies (Goethals et al., 1992; Kohler et al., 1997; 1999; Maseda et al., 2010; Tian et al., 2009). We identified MexT-binding sequence with the upstream region of the
Since recombinant MexT was not tested with candidate ligands, its binding to MexT binding sites I and II likely represents its inactive status as a
In conclusion, MexT is a LTTR (Kohler et al., 1997; 1999). The typical tetrameric arrangement of OxyR (Jo et al., 2015) and the atypical tetrameric arrangement of VV2_1132 and HypT have been characterized previously (Jang et al., 2018; Jo et al., 2019). Recognition of ligand binding or stimuli by the RD induces structural changes in transcriptional regulators, which alters their DNA binding (Jo et al., 2015). We elucidated the crystal structure of the RD of MexT of
This work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1A2B2003992 to NCH). We made use of beamline 5C at the Pohang Accelerator Laboratory (Pohang, Republic of Korea).
Statistics for X-ray data collection and refinement
Native MexT RD | Se-Met MexT RD | |
---|---|---|
Data collection | ||
Beamline | PAL 5C | PAL 5C |
Wavelength (Å) | 0.97960 | 0.97940 |
Space group | ||
Cell dimensions | ||
| 65.7, 108.7, 109.2 | 65.9, 108.7, 109.3 |
α, β, γ (°) | 90, 90, 90 | 90, 90, 90 |
Resolution (Å) | 50-2.00 (2.03-2.00)a | 50-2.30 (2.34-2.30)a |
| 0.042 (0.50) | 0.10 (0.83) |
| 23.76 (2.91) | 13.07 (2.08) |
Completeness (%) | 97.1 (93.6) | 98.8 (94.4) |
Redundancy | 5.6 (3.7) | 7.9 (3.5) |
Refinement (PDB code : 6L33) | ||
Resolution (Å) | 31.8-2.0 | |
No. of reflections | 50,630 | |
| 0.21/0.25 | |
No. of total atoms | 6,365 | |
Wilson B-factor (Å) | 22.18 | |
RMSD | ||
Bond lengths (Å) | 0.003 | |
Bond angles (°) | 0.67 | |
Ramachandran plot | ||
Favored (%) | 97.0 | |
Allowed (%) | 3.0 | |
Outliers (%) | 0.0 | |
PDB ID | 6L33 |
RMSD, root mean square deviation.
b
c
Mol. Cells 2019; 42(12): 850-857
Published online December 31, 2019 https://doi.org/10.14348/molcells.2019.0168
Copyright © The Korean Society for Molecular and Cellular Biology.
Suhyeon Kim1, Songhee H. Kim2, Jinsook Ahn1, Inseong Jo1,3, Zee-Won Lee1, Sang Ho Choi1, and NamChul Ha1,*
1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea, 2Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Korea, 3Present address: KoBioLabs, Inc., Seoul 08826, Korea
Correspondence to:hanc210@snu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The Gram-negative opportunistic pathogen,
Keywords: antibiotic resistance, crystal structure, lysR-type transcriptional regulator, MexT, Pseudomonas aeruginosa
The opportunistic human pathogen
The expression level of MexEF-OprN in wild-type strains under laboratory conditions is typically negligible. However, treatment with the fluoroquinolone antibiotic norfloxacin leads to generate certain types of spontaneous mutant strains, such as
We conducted the experiments to evaluate the molecular mechanism of MexT by understanding its structure. We investigated the mechanism by which MexT regulates the expression of MexEF-OprN by producing the full-length (FL) recombinant protein in
The native MexT RD (residues 95–304) and FL MexT (residues 1–304) were expressed as described previously (Kim et al., 2018). For the selenomethionyl (SeMet)-labeled MexT RD, the host
After TALON affinity chromatography, FL MexT was directly loaded onto a size-exclusion chromatography column (HiLoad Superdex 16/60 200; GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 10% (v/v) glycerol, and 2 mM 2-mercaptoethanol. The purified proteins were aliquoted and stored frozen at −173°C.
MexT RD was crystallized using the hanging-drop vapor-diffusion method at 14°C as described previously (Kim et al., 2018). The precipitation solution comprised 0.5 M ammonium sulfate, 1.0 M sodium citrate tribasic dehydrate (pH 5.6), 0.1 M lithium sulfate, and 2 mM ethylenediaminetetraacetic acid (EDTA). Glycerol (30% [v/v], cryoprotectant) was added to the precipitation solution. The X-ray diffraction data were collected in a flash-cooled liquid nitrogen stream at −173°C. SeMet-MexT RD was crystallized under the same crystallization conditions as for native MexT RD. The single-wavelength anomalous diffraction (SAD) dataset was collected from beamline 5C at the Pohang Accelerator Laboratory (Korea) and processed using HKL2000 software (Otwinowski and Minor, 1997).
The 2.0 Å resolution dataset of the native crystal belongs to space group
The sizes of FL MexT and MexT RD were assayed using SEC-MALS. A high-performance liquid chromatography pump (Agilent, USA) was connected to a Superdex-200 10/300 GL gel filtration column (GE Healthcare) and a MALS instrument (Wyatt Dawn Heleos, USA). The size-exclusion chromatography column was pre-equilibrated with buffer comprising 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 2 mM 2-mercaptoethanol for FL MexT; and 20 mM Tris-HCl (pH 8.5), 300 mM NaCl, and 2 mM 2-mercaptoethanol for MexT RD. Bovine serum albumin (2 mg/ml) was used as the standard. MexT RD and FL MexT samples (2 mg/ml) were injected onto the column and eluted at a flow rate of 0.2 ml/min. The datasets were evaluated using the Debye model for fitting static light-scattering data, and refractive index peaks were presented in EASI graphs created using Astra V software (Wyatt Dawn Heleos).
A DNA fragment of 630 bp (the
The PCR product was purified from agarose gel electrophoresis. We mixed 400 ng of DNA and 200 ng of purified FL MexT for 30 min at room temperature. Next, 0.04 units of DNase I (Fermentas) was added, and the reaction mixture (10 mM Tris [pH 8.0], 50 mM KCl, 8 mM MgCl2, 50 ng/μl BSA, 5% glycerol) was incubated for 2 min at room temperature. The reactions were terminated by adding an equal volume of stopping buffer (200 mM NaCl, 1% sodium dodecyl sulfate, 30 mM EDTA), and DNase I was inactivated by heating at 75°C for 10 min. The DNA fragments were recovered via phenol extraction and alcohol precipitation and fragment-sequences were analyzed using an ABI3730xl DNA Analyzer (Applied Biosystems, USA). The size distribution was scanned using Peak Scanner software (ver. 1.0; Applied Biosystems).
The FL MexT (residues 1–304) and its RD (residues 95–304) were produced in
To obtain phase information, we acquired the diffraction dataset using the crystal of the SeMet-labeled protein, which was crystallized in the presence of a high concentration of ammonium sulfate. The structure was determined via the SAD method using the anomalous signals from SeMet, which was refined to the 2.0-Å-resolution native dataset with an
MexT RD protomers are composed of 10 β-strands and 7 α-helices, as in the typical LTTR RD (Jo et al., 2015). Each protomer can be divided into two subdomains—RD-I (residues 95–172, 274–304) and RD-II (residues 173–273). The folding pattern of the two subdomains was similar to Rossman-fold topology; i.e., a central β-sheet bound by helices and loops. The RD-I subdomain comprised three α helices (α1, α2, and α7) and five β-strands (β1, β2, β3, β4, and β10), whereas the RD-II subdomain comprised four α helices (α3, α4, α5, and α6) and five β-strands (β5, β6, β7, β8, and β9). The RD-I and RD-II subdomains were connected by two loops—one connects β4 of RD-I to β5 of RD-II, and the other connects β9 of RD-II to β10 of RD-I (Fig. 2).
The four protomers (chains A–D) in the asymmetric unit are arranged as two of dimers (chains A:B and C:D; Fig. 2). The overall arrangement of the MexT RD dimer was similar to those of other LTTRs. Within crisscrossing protomer dimer, RD-I of one protomer directly interacted with RD-II of the other protomer. The major interactions of two protomers were intermolecular hydrogen bonds between residues in the RD-1 β2 backbone and the RD-II β7 backbone at the dimeric interface. Hydrophobic interactions were found at the dimeric interface of RD-1 α1 and RD-II α5, and likely stabilize the dimer (Supplementary Fig. S1).
The asymmetric unit contained five sulfate ions, which may originate from the crystallization solution (Fig. 3 and Supplementary Fig. S2). Of the five sulfate ions, three were found at the dimeric interfaces; the sulfate ions interacted with Arg134 and Arg135 near the molecular pseudo-2-fold axis—one in the A:B chain dimer and two in the C:D chain dimer (Fig. 3). The fourth sulfate ion is found in the putative ligand-binding site (Fig. 4A). The fifth sulfate ion is located outside of the A:B chain dimer, interacting with Arg120 and Arg121 of chain A (Supplementary Fig. S2).
The dimeric arrangements of the two dimers were similar (Supplementary Fig. S3), but the relative position of subdomain RD-II within the overall frame showed ~5.0 Å deviation. The orientation of the protomers in the dimer can be used to distinguish active from inactive LTTRs (Choi et al., 2001; Jo et al., 2015). To examine the conformation of the RD dimer, the obtained MexT RD dimer (chains A:B) was superimposed onto the
Compared to a typical LTTR
As mentioned above, one sulfate ion is located at the putative ligand-binding site (Fig. 3A). Arg220 and Arg269 ionically interact with the sulfate ion at the putative ligand-binding site of chain B (Fig. 3A). In particular, guanidine group of Arg269 in chain B can make two configurations by flipping. And it is the site where one sulfate ion can bind. Pocket-formation by the flipping guanidine group makes space for holding small molecules. The sulfate-binding region extends to the hydrophobic surface region decorated by Tyr138, Phe201, Phe208, and Phe229 (Fig. 4B and Supplementary Fig. S5). Highlighting the space of adjacent sulfate-binding region and hydrophobic patches in the ligand-binding site, our structure suggests that candidate ligands should have a negatively charged (or polar) moiety linked to a hydrophobic moiety.
Cinnamaldehyde upregulated the expression of MexEF-OprN. Indeed, cinnamaldehyde has a polar aldehyde moiety and a hydrophobic phenyl ring (Juarez et al., 2017). The polar aldehyde moiety likely interacts with the positively charged pocket, which is composed of Arg220 and Arg269, and the hydrophobic phenyl ring probably participates in the interaction. A molecular model showed that cinnamaldehyde fitted the pocket well (Dallakyan and Olson, 2015) (Supplementary Fig. S6). Molecules with such characteristics may activate MexT and alter its association with DNA.
To elucidate the interaction between MexT with the promoter of
Next, the two MexT-binding site sequences identified by DNase I footprinting were aligned with the promoter sequences of other genes in the MexT regulon (PA1744, PA1970, PA2486, and PA2759). The MexT regulon was identified with microarray-based transcriptome profiling (Tian et al., 2009), and was further experimentally confirmed by electromobility shift assays and β-galactosidase assays using transcriptional fusion reporters (Maseda et al., 2010; Tian et al., 2009). The sequence alignment showed a palindromic consensus pattern of ATCA(N7)CGAT at each MexT-binding site. Notably, there was partial overlap between the front region
The MexT-binding sequence has been predicted in previous studies (Goethals et al., 1992; Kohler et al., 1997; 1999; Maseda et al., 2010; Tian et al., 2009). We identified MexT-binding sequence with the upstream region of the
Since recombinant MexT was not tested with candidate ligands, its binding to MexT binding sites I and II likely represents its inactive status as a
In conclusion, MexT is a LTTR (Kohler et al., 1997; 1999). The typical tetrameric arrangement of OxyR (Jo et al., 2015) and the atypical tetrameric arrangement of VV2_1132 and HypT have been characterized previously (Jang et al., 2018; Jo et al., 2019). Recognition of ligand binding or stimuli by the RD induces structural changes in transcriptional regulators, which alters their DNA binding (Jo et al., 2015). We elucidated the crystal structure of the RD of MexT of
This work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1A2B2003992 to NCH). We made use of beamline 5C at the Pohang Accelerator Laboratory (Pohang, Republic of Korea).
. Statistics for X-ray data collection and refinement.
Native MexT RD | Se-Met MexT RD | |
---|---|---|
Data collection | ||
Beamline | PAL 5C | PAL 5C |
Wavelength (Å) | 0.97960 | 0.97940 |
Space group | ||
Cell dimensions | ||
| 65.7, 108.7, 109.2 | 65.9, 108.7, 109.3 |
α, β, γ (°) | 90, 90, 90 | 90, 90, 90 |
Resolution (Å) | 50-2.00 (2.03-2.00)a | 50-2.30 (2.34-2.30)a |
| 0.042 (0.50) | 0.10 (0.83) |
| 23.76 (2.91) | 13.07 (2.08) |
Completeness (%) | 97.1 (93.6) | 98.8 (94.4) |
Redundancy | 5.6 (3.7) | 7.9 (3.5) |
Refinement (PDB code : 6L33) | ||
Resolution (Å) | 31.8-2.0 | |
No. of reflections | 50,630 | |
| 0.21/0.25 | |
No. of total atoms | 6,365 | |
Wilson B-factor (Å) | 22.18 | |
RMSD | ||
Bond lengths (Å) | 0.003 | |
Bond angles (°) | 0.67 | |
Ramachandran plot | ||
Favored (%) | 97.0 | |
Allowed (%) | 3.0 | |
Outliers (%) | 0.0 | |
PDB ID | 6L33 |
RMSD, root mean square deviation..
b
c
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