The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1
Abstract
:1. Introduction
2. Results
2.1. Structure of EppA
2.2. Isothermal Titration Calorimetry
2.3. Molecular Docking
2.4. The Electrostatic Potentials of the Ligand-Binding Pocket
2.5. Site-Directed Mutagenetic Analysis of Key Binding Residues
2.6. Structural Homologs of EppA and Evolutionary Conservation
3. Discussion
4. Materials and Methods
4.1. Site-Directed Mutagenesis
4.2. Protein Expression and Purification
4.3. Molecular Mass Determination
4.4. Crystallization
4.5. Structure Determination
4.6. Structure Analysis
4.7. Isothermal Titration Calorimetry
4.8. Quantum Chemical Optimization and Electrostatics
4.9. Molecular Docking
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Kd (μM) | ∆H (kcal mol−1) | ∆S (cal mol−1 K−1) | |
---|---|---|---|
Asp10 b | n.b. c | n.b. | n.b. |
EDDA d | 0.340 ± 0.118 | −0.851 ± 0.893 | 26.7 |
EDTA | 0.0095 ± 0.006 | −2.283 ± 0.07 | 29 |
EGTA | 0.169 ± 0.71 | −4.34 ± 0.114 | 16.4 |
MgEDTA | 0.315 ± 3.26 | −4.028 ± 0.037 | 16.2 |
CaEDTA | 2.320 ± 20.41 | −2.917 ± 0.038 | 16.0 |
SrEDTA | 1.69 ± 7.69 | −4.392 ± 0.118 | 27.1 |
BaEDTA | n.b. | n.b. | n.b. |
CrEDTA | 0.019 ± 0.002 | −2.573 ± 0.145 | 30.5 |
MnEDTA | n.b. | n.b. | n.b. |
FeEDTA | n.b. | n.b. | n.b. |
CoEDTA | n.b. | n.b. | n.b. |
NiEDTA | n.b. | n.b. | n.b. |
CuEDTA | n.b. | n.b. | n.b. |
ZnEDTA | n.b. | n.b. | n.b. |
CdEDTA | 0.28 ± 0.49 | −0.555 ± 0.002 | 28.1 |
PrEDTA | 6.17 ± 55.25 | −5.366 ± 0.133 | 5.83 |
NdEDTA | 8.85 ± 141.2 | −4.889 ± 0.063 | 6.73 |
EuEDTA | 26.45 ± 363.6 | −3.943 ± 0.105 | 7.72 |
TbEDTA | 31.25 ± 116.4 | −0.751 ± 0.069 | 18.1 |
MgEGTA | 0.315 ± 3.26 | −4.028 ± 0.037 | 16.2 |
CaEGTA | n.b. | n.b. | n.b. |
SrEGTA | 4.50 ± 10.5 | 0.794 ± 0.164 | 27.1 |
BaEGTA | n.b. | n.b. | n.b. |
Ligand * | ΔGbinding,A (kcal mol−1) | ΔGbinding,B (kcal mol−1) |
---|---|---|
EDTA | −6.4 | −6.6 |
EGTA | −6.0 | −6.1 |
NTA | −5.2 | −6.5 |
EDDA | −5.2 | −4.9 |
HEDTA | −6.0 | −6.1 |
EDDS | −6.1 | −6.7 |
DTPA | −6.6 | −6.7 |
EDDHA | −7.0 | −7.0 |
BAPTA | −6.9 | −6.9 |
Kd (nM) | ∆H (kcal mol−1) | ∆S (cal mol−1 K−1) | |
---|---|---|---|
Wild-type | 9.52 ± 6.25 | −2.283 ± 0.070 | 29 |
R56A | 161 ± 272 | −1.802 ± 0.147 | 25 |
R74A | 249 ± 382 | −1.877 ± 0.195 | 23.9 |
K470A | 27.1 ± 20.2 | −1.872 ± 0.117 | 28.3 |
R480A | 40.8 ± 26.5 | −1.138 ± 0.089 | 30 |
Protein | PDB ID | Source | % Identity | % Similarity | Z-Score |
---|---|---|---|---|---|
LpqW | 2GRV | Mycobacterium smegmatis | 22.5 | 38.8 | 29.0 |
CBP | 2O7I | Thermotoga maritima | 19.6 | 33.5 | 24.4 |
AgaA | 6HLX | Rhizobium radiobacter | 18.8 | 35.7 | 31.4 |
OppA | 3DRF | Lactococcus lactis | 18.7 | 35.2 | 27.6 |
OppA2 | 2WOK | Streptomyces clavuligerus | 18.3 | 32.3 | 29.1 |
MnBP3 | 4PFT | Thermotoga maritima | 18.3 | 35.3 | 27.6 |
MoaA | 6TFX | Rhizobium radiobacter | 18.2 | 33.5 | 30.3 |
NikZ | 4OET | Campylobacter jejuni | 18.1 | 36.8 | 34.4 |
CtaP | 5ISU | Listeria monocytogenes | 18.0 | 33.9 | 34.2 |
CosBP | 1ZTY | Vibrio cholerae | 17.9 | 33.3 | 21.0 |
EppA (apo) | EppA (EDTA Soak) | |
---|---|---|
Data collection | ||
Space group | P43212 | P212121 |
Cell dimensions | ||
a, b, c (Å) | 87.81, 87.81, 164.48 | 84.34, 90.61, 165.27 |
α, β, γ (°) | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 |
Resolution (Å) | 49.46—1.42 (1.47—1.42) | 47.07—1.56 (1.62—1.56) |
Rmerge | 0.16 (0.860) | 0.125 (1.59) |
Wavelength (Å) | 0.9793 | 1.000 |
Unique reflections | 122,095 (11,723) | 179,416 (17,354) |
Completeness (%) | 97.40 (89.55) | 94.44 (77.22) |
<I>/σI | 10.57 (4.77) | 12.65 (1.22) |
CC1/2 | 0.996 (0.887) | 0.999 (0.375) |
CC * | 0.999 (0.97) | 1 (0.738) |
Multiplicity | 20.5 (16.6) | 13.5 (6.2) |
Refinement | ||
Rwork/Rfree | 0.145 (0.187)/0.153 (0.210) | 0.153 (0.310)/0.169 (0.317) |
CCwork/CCfree | 0.971 (0.919)/0.966 (0.915) | 0.968 (0.709)/0.966 (0.691) |
Number of atoms | ||
Protein | 4209 | 8310 |
Sulfate | 20 | 15 |
Ethylene glycol | 63 | 80 |
Water | 604 | 1285 |
ADP (Å2) | ||
All atoms | 26.05 | 21.33 |
Protein | 24.45 | 19.57 |
Ligands | 34.5 | 30.2 |
Solvent | 36.58 | 32.04 |
R.m.s deviations | ||
Bonds (Å) | 0.008 | 0.008 |
Angles (°) | 1.31 | 1.27 |
Ramachandrans | ||
% Favored | 98.31 | 98.12 |
% Outliers | 0 | 0 |
Rotamer outliers | 0.45 | 0.23 |
Clashscore | 1.79 | 2.29 |
TLS groups | 3 | 6 |
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Lewis, K.M.; Greene, C.L.; Sattler, S.A.; Youn, B.; Xun, L.; Kang, C. The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1. Int. J. Mol. Sci. 2020, 21, 3940. https://doi.org/10.3390/ijms21113940
Lewis KM, Greene CL, Sattler SA, Youn B, Xun L, Kang C. The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1. International Journal of Molecular Sciences. 2020; 21(11):3940. https://doi.org/10.3390/ijms21113940
Chicago/Turabian StyleLewis, Kevin M., Chelsie L. Greene, Steven A. Sattler, Buhyun Youn, Luying Xun, and ChulHee Kang. 2020. "The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1" International Journal of Molecular Sciences 21, no. 11: 3940. https://doi.org/10.3390/ijms21113940