Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells
Abstract
:1. Introduction
2. MaterialsandMethods
2.1. Bacterial Strains
2.2. MolecularCloning
2.3. Expression and Purification of the Recombinant Protein
2.4. Substrate Specificity Assay
2.5. Activity Assay
2.6. Protein Crystallization, Data Collection and Processing, Structure Solution
3. Results
3.1. Bioinformatic Analysis
3.2. Molecular Cloning and the Properties of Recombinant AcLys
3.3. Substrate Specificity Assay
3.4. Activityand Optimal Enzymologic Conditions of AcLys
3.5. Oligomeric State of AcLys
3.6. Stability of AcLys Protein
3.7. Structural Analysis
3.8. Comparison to Known Structures
4. Discussion
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Bacterium | Strain | Apparent MIC *, 2 h in LB, µg/mL |
---|---|---|
Acinetobacter baumannii | ATCC19606 | 100 |
Acinetobacter baumannii | AB5075 | 100 |
Acinetobacter pitii | ATCC17978 | 100 |
Escherichia coli | CF1073 | 50 |
Escherichia coli | CR63 | 50 |
Klebsiella pneumoniae | GISK 233 | 50 |
Pseudomonas aeruginosa | PAO1 | 50 |
Proteus vulgaris | GISK 185 | 100 |
Bacillus thuringiensis | GISK 202-E | - |
Staphylococcus aureus | GISK 222 | - |
Data Collection | Refinement | ||
---|---|---|---|
Space group | P22121 | Rwork/Rfree | 14.7/16.9 |
Cell dimensions | No. of atoms | ||
a, b, c (Å) | 30.98; 67.00; 75.76 | Protein | 1152 |
α, β, γ (°) | 90; 90; 90 | Ligands/ion | 11 |
Resolution (Å) | 50.19–1.20 (1.22–1.20) | Water | 229 |
Rmeas (%) | 12.4 (100.0) | Ramachandran outliers, % | 0 |
CC1/2 | 99.4 (77.7) | Ramachandran favored, % | 98 |
<I>/<σ(I)> | 5.6 (1.5) | R.m.s deviations | |
Completeness | 94.0 (94.5) | Bond length (Å) | 0.019 |
Redundancy | 5.0 (5.0) | Bond angle (°) | 0.980 |
MolProbity Score | 1.44 |
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Sykilinda, N.N.; Nikolaeva, A.Y.; Shneider, M.M.; Mishkin, D.V.; Patutin, A.A.; Popov, V.O.; Boyko, K.M.; Klyachko, N.L.; Miroshnikov, K.A. Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells. Viruses 2018, 10, 309. https://doi.org/10.3390/v10060309
Sykilinda NN, Nikolaeva AY, Shneider MM, Mishkin DV, Patutin AA, Popov VO, Boyko KM, Klyachko NL, Miroshnikov KA. Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells. Viruses. 2018; 10(6):309. https://doi.org/10.3390/v10060309
Chicago/Turabian StyleSykilinda, Nina N., Alena Y. Nikolaeva, Mikhail M. Shneider, Dmitry V. Mishkin, Artem A. Patutin, Vladimir O. Popov, Konstantin M. Boyko, Natalia L. Klyachko, and Konstantin A. Miroshnikov. 2018. "Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells" Viruses 10, no. 6: 309. https://doi.org/10.3390/v10060309