Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning of the GST-PupB NTSD and MBP-PupR CCSSD and Mutagenesis
2.2. Expression and Purification of PupB NTSD and Mutants
2.3. Quantification of the MBP-PupR CCSSD Interaction with PupB NTSD Mutants
2.4. CD Spectroscopy and Thermal Denaturation of PupB NTSD Mutants
2.5. NMR Spectroscopy
2.5.1. NMR Sample Preparation, Data Collection, and Chemical Shift Assignment
2.5.2. NMR Hydrogen/Deuterium Exchange Experiments
2.5.3. Hydrogen Bond Restraints
2.5.4. Dihedral Angle Restraints
2.5.5. NOE Assignment and Structure Calculation
2.5.6. Structure Validation
2.5.7. 1H–15N HSQC Spectroscopy of Labelled PupB NTSD Mutants
3. Results
3.1. Solution NMR Structure of the PupB NTSD
3.2. Effect of Mutations on Interaction between the NTSD and the CCSSD
3.3. Effect of NTSD Mutations on Folding and Thermal Stability
3.4. Comparison of 1H–15N HSQC Mutant and WT Spectra Indicates the Structural Effects of Mutation Are Modest
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
List of Abbreviations
Cell surface signaling | CSS |
C-terminal cell surface signaling domain | CCSSD |
Circular dichroism spectroscopy | CD |
Ferric citrate | Fec |
Ferric pyoverdine | Fpv |
Isothermal titration calorimetry | ITC |
N-terminal signaling domain | NTSD |
Nuclear magnetic resonance | NMR |
Pseudobactin BN7/8 uptake | Pup |
Wild-type | WT |
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List of Constraints | |
---|---|
NOE Distance Restraints: | |
Unambiguous | 1312 |
Ambiguous | 181 |
Hydrogen Bond Restraints | 50 |
Dihedral Angle Restraints | 144 |
Structural Analysis | |
Mean r.m.s.d. from experimental restraints: | |
NOE (Å) | 0.501 ± 0.031 |
Dihedral angles (deg.) | 5.834 ± 0.927 |
Average number of: | |
NOE violations > 0.5 Å | 70.3 ± 2.4 |
NOE violations > 0.3 Å | 99.9 ± 4.3 |
Dihedral violations > 5° | 12.4 ± 1.7 |
Mean rms from idealized covalent geometry: | |
Bonds, Å | 0.0099 ± 0.0004 |
Angles, ° | 1.06 ± 0.03 |
Impropers, ° | 1.28 ± 0.06 |
Geometric analysis of residues 2–82 | |
Rmsd to mean, backbone, Å | 0.79 ± 0.35 |
Rmsd to mean, all heavy, Å | 1.46 ± 0.34 |
Ramachandran Analysis (Procheck) | |
Most-favored region (%) | 62.5 |
Additionally Allowed region (%) | 33.4 |
Generously Allowed region (%) | 3.9 |
Disallowed region (%) | 0.15 |
PupB NTSD | Kd (µM) | ΔH (kJ/mol) | ΔS (J/mol·K) | ΔG (kJ/mol) | Incompetent Fraction (%) |
---|---|---|---|---|---|
WT | 0.69 [0.42, 1.11] a | −73.99 [−80.99, −68.27] a | −138.83 [−158.94, −122.93] a | −33.99 [−35.18, −32.84] a | 0.0–12.8% |
Q69K | 1.55 [0.9, 2.6] a | −60.70 [−67.83, −55.03] a | −99.42 [−119.81, −84.05] a | −32.05 [−33.31, −30.81] a | 0.0–9.3% |
H72D | 27.71 [U b, 125.63] a | −96.16 [U b, −67.65] a | −246.46 [U b, −122.93] a | −25.14 [U b, −21.52] a | 0.0–43.5% |
L74A | No Interaction Detected | - | - | - | - |
PupB NTSD | Helix | Strand | Coil + Turn | Total | Tm (Heating) | Tm (Cooling) |
---|---|---|---|---|---|---|
WT | 19.5 ± 1.2 | 36.6 ± 1.2 | 43.9 ± 0 | 100 | 57.4 ± 0.1 °C | 53.8 ± 0.9 °C |
Q69K | 12.2 ± 3.7 | 37.8 ± 4.9 | 50.0 ± 1.2 | 100 | 57.8 ± 0.9 °C | 58.9 ± 0.1 °C |
H72D | 11.0 ± 3.7 | 37.8 ± 2.4 | 51.2 ± 1.2 | 100 | 53.1 ± 0.3 °C | 53.3 ± 0.3 °C |
L74A | 20.7 ± 1.2 | 36.6 ± 1.2 | 42.7 ± 1.2 | 100 | 56.3 ± 0.2 °C | 56.3 ± 0.2 °C |
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Sultana, T.; Morgan, D.M.; Jernberg, B.D.; Zak, P.; Sinha, S.C.; Colbert, C.L. Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum. Biomolecules 2024, 14, 1108. https://doi.org/10.3390/biom14091108
Sultana T, Morgan DM, Jernberg BD, Zak P, Sinha SC, Colbert CL. Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum. Biomolecules. 2024; 14(9):1108. https://doi.org/10.3390/biom14091108
Chicago/Turabian StyleSultana, Tajnin, David M. Morgan, Beau D. Jernberg, Peyton Zak, Sangita C. Sinha, and Christopher L. Colbert. 2024. "Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum" Biomolecules 14, no. 9: 1108. https://doi.org/10.3390/biom14091108