The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bioinformatic Analysis
2.2. Expression and Purification of LysF1
2.3. Turbidity Reduction Assay
2.4. Differential Scanning Fluorimetry
2.5. X-ray Crystallography
2.6. Analytical Ultracentrifugation
2.7. Small-Angle X-ray Scattering
2.8. Circular Dichroism
3. Results and Discussion
3.1. Protein Sequence Analysis Suggests LysF1 Is a Glycoside Hydrolase Family 24 Member
3.2. LysF1 Is an Endolysin That Causes Lysis of Outer Membrane-Permeabilized Gram-Negative Bacteria
3.3. The Structure of LysF1
3.4. The Monomeric LysF1 Solution Structure Is Consistent with the Crystal Structure
3.5. LysF1 Catalyzes Peptidoglycan Degradation in a T4L-like Mechanism
3.6. LysF1 Retains T4L-Like Architecture with Context Important Mutations
3.7. Improving the Function and Stability of LysF1 Through Mutation of the Hydrophobic Core
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Data Collection Statistics | |
Wavelength (Å) | 0.953719 |
Temperature (K) | 100 |
Detector | EIGER X 16M (Dectris) |
Space group | P 32 2 1 |
Unit cell parameters (Å) | a = 67.87, b = 67.87, c = 134.44 |
Resolution range (Å) | 44.81–1.71 (1.74–1.71) |
Observed reflections | 329,379 (15,760) |
Unique reflections | 39,743 (2042) |
Mean I/σ(I) | 13.1 (1.2) |
CC1/2 | 0.999 (0.531) |
Completeness (%) | 99.8 (96.1) |
Rmerge | 0.074 (1.645) |
Rr.i.m. | 0.079 (1.761) |
Rp.i.m. | 0.028 (0.619) |
Wilson B value (Å2) | 29.9 |
VM (Å3 Da−1) | 2.30 |
Molecules per asymmetric unit | 2 |
Solvent content (%) | 46 |
Structure and Refinement Statistics | |
Rfactor (%) | 18.4 |
Rfree (%) | 20.7 |
Number of atoms | |
Macromolecules | 2483 |
Water | 299 |
Ligands | 20 |
Root-mean-square deviation (r.m.s.d.) | |
Bonds (Å) | 0.01 |
Angles (°) | 1.8 |
Average B factors (Å2) | |
Macromolecules | 34.8 |
Water | 43.6 |
Ligands | 59.7 |
Ramachandran plot, residues (%) | |
Favored region | 99 |
Allowed region | 1 |
Disallowed region | 0 |
PDB ID | 7M5I |
SAXS Data Analysis | |
---|---|
I(0) (cm−1) | 0.021 |
Rg (Å) | 18.53 |
P(r) analysis | |
I(0) (cm−1) | 0.02 |
Rg (Å) | 18.95 |
Dmax (Å) | 68.39 |
Porod volume (Å−3) | 27,014.90 |
Molar mass from Porod Volume (Da) | 15,475 |
Calculated monomeric from sequence a (Da) | 19,575.25 |
Endolysin | Putative Key Catalytic Residues | PDB ID | Reference | ||
---|---|---|---|---|---|
LysF1 | Glu15 | Asp24 | Thr30 | 7M5I | This work |
T4L | Glu11 | Asp20 | Thr26 | 2LZM | [51] |
P1 endolysin | Glu42 | Cys51 | Thr57 | 1XJT | [50] |
P22 endolysin | Glu16 | Asp25 | Thr31 | 2ANX | [48] |
P21 endolysin | Glu35 | Asp44 | Thr50 | 3HDE | [49] |
DLP12 endolysin | Glu35 | Asp44 | Thr50 | 4ZPU | [47] |
AcLys | Glu64 | Asp73 | Thr79 | 6ET6 | [46] |
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Love, M.J.; Coombes, D.; Manners, S.H.; Abeysekera, G.S.; Billington, C.; Dobson, R.C.J. The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability. Viruses 2021, 13, 1101. https://doi.org/10.3390/v13061101
Love MJ, Coombes D, Manners SH, Abeysekera GS, Billington C, Dobson RCJ. The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability. Viruses. 2021; 13(6):1101. https://doi.org/10.3390/v13061101
Chicago/Turabian StyleLove, Michael J., David Coombes, Sarah H. Manners, Gayan S. Abeysekera, Craig Billington, and Renwick C. J. Dobson. 2021. "The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability" Viruses 13, no. 6: 1101. https://doi.org/10.3390/v13061101