Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmids
2.2. Protein Expression and Protein Purification
2.3. NLS Peptides
2.4. Crystallization and Data Processing
2.5. Fluorescence Polarization
2.6. EMSA
2.7. Co-Immunoprecipitation (Co-IP) Assay
2.8. Western Blot
2.9. Immunofluorescence Assays
3. Results
3.1. Henipavirus M NLS1 Binds to IMPα at the Major Site
3.2. M NLS2 Binds to IMPα at the Minor Site
3.3. M NLS1 and NLS2 Bind IMPα Isoforms in Electromobility Shift Assays and Fluorescence Polarization Assays
3.4. The M NLS2 Is an Important Binding Interface in a Cellular Context
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Data Collection and Processing | IMPα2:HeV M NLS1 | IMPα2:HeV M NLS2 | IMPα3:HeV M NLS1 |
---|---|---|---|
Wavelength (Å) | 0.9537 | 0.9537 | 0.9537 |
Resolution range (Å) | 24.42–1.9 (1.94–1.9) | 19.78–2.10 (2.16–2.10) | 29.78–2.75 (2.9–2.75) |
Space group | P 21 21 21 | P 21 21 21 | P 21 21 21 |
Unit cell (Å, o) | 78.49 89.86 99.83 90 90 90 | 78.08 89.50 97.06, 90 90 90 | 49.06 64.27 158.58 90 90 90 |
Total reflections | 405,486 (26,486) | 167,351 (12,946) | 154,985 (23,139) |
Unique reflections | 56,298 (3749) | 40,357 (3253) | 13,681 (1966) |
Multiplicity | 7.2 (7.1) | 4.1 (4.0) | (11.8) |
Completeness (%) | 99.9 (100) | 99.9 (99.9) | 99.9 (100) |
Mean I/sigma (I) | 11.9 (1.5) | 11.0 (1.9) | 7.8 (2.3) |
Wilson B-factor Å2 | 29.65 | 35.12 | 55.58 |
R-merge | 0.085 (1.376) | 0.063 (0.696) | 0.181 (1.032) |
R-pim | 0.050 (0.820) | 0.035 (0.400) | 0.078 (0.446) |
CC1/2 | 0.998 (0.602 | 0.998 (0.734) | 0.995 (0.887) |
Refinement | |||
Number of reflections | 56,237 (5567) | 40,289 (3941) | 13,635 (1323) |
Number of R-free reflections | 2864 (322)) | 1981(180) | 671 (80) |
R-work % | 0.1728 (0.2853) | 0.1791 (0.2520) | 0.2411 (0.3185) |
R-free % | 0.1900 (0.3147) | 0.2053 (0.3058) | 0.2811 (0.3553) |
RMS (bonds) | 0.015 | 0.006 | 0.003 |
RMS (angles) | 1.18 | 0.77 | 0.58 |
Ramachandran | |||
favored (%) | 98.6 | 98.14 | 97.85 |
allowed (%) | 1.4 | 1.86 | 2.15 |
outliers (%) | 0.00 | 0.00 | 0.00 |
Average B-factor Å2 | 43.57 | 50.33 | 72.53 |
Clash score | 1.54 | 4.07 | 4.92 |
PDB code | 8FUA | 8FUC | 8FUB |
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Donnelly, C.M.; Vogel, O.A.; Edwards, M.R.; Taylor, P.E.; Roby, J.A.; Forwood, J.K.; Basler, C.F. Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism. Viruses 2023, 15, 1302. https://doi.org/10.3390/v15061302
Donnelly CM, Vogel OA, Edwards MR, Taylor PE, Roby JA, Forwood JK, Basler CF. Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism. Viruses. 2023; 15(6):1302. https://doi.org/10.3390/v15061302
Chicago/Turabian StyleDonnelly, Camilla M., Olivia A. Vogel, Megan R. Edwards, Paige E. Taylor, Justin A. Roby, Jade K. Forwood, and Christopher F. Basler. 2023. "Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism" Viruses 15, no. 6: 1302. https://doi.org/10.3390/v15061302
APA StyleDonnelly, C. M., Vogel, O. A., Edwards, M. R., Taylor, P. E., Roby, J. A., Forwood, J. K., & Basler, C. F. (2023). Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism. Viruses, 15(6), 1302. https://doi.org/10.3390/v15061302