| _version_ | 1866901823631130624 |
|---|---|
| author | Pustovalova, Yulia |
| author_facet | Pustovalova, Yulia |
| contents | <h3><strong>Abstract</strong></h3> <p>Positive-strand RNA viruses remodel host intracellular membranes to form replication organelles in cytoplasm. SARS-CoV-2 and other coronaviruses, which belong to <em>Nidovirales </em>order, replicate within double-membrane vesicles (DMVs) derived from the endoplasmic reticulum that protect newly synthetized viral RNA (vRNA). The vRNA must then be exported to cytoplasm for packaging into virions. A recently discovered SARS-CoV-2 molecular pore complex composed of non-structural proteins 3 and 4 (nsp3 and nsp4) connects the DMV lumen with the cytoplasm, enabling vRNA export. The C-terminal region of nsp3, the Y domain, oligomerizes to form the base of the cytosolic crown of this pore, while preceding transmembrane domain 2 (TM2) anchors the pore into DMV membrane. However, the molecular mechanisms underlying pore formation and whether similar structures are conserved across other <em>Nidovirales </em>members remain unclear. Here, we combined AlphaFold structure prediction, structure-informed multiple sequence alignments, and atomistic molecular dynamics simulations to investigate the conservation and potential function of C-terminal TM2-Y region of nsp3 across nidoviruses. We show that the wedge-shaped TM2, Zn-binding Y1 subdomain, and its adjacent Y2 subdomain constitute a conserved structural module present in most vertebrate-infecting <em>Nidovirales</em> families, including <em>Coronaviridae</em>, <em>Tobaniviridae</em>, <em>Gresnaviridae</em>, <em>Olifoviridae</em> and <em>Arteriviridae</em>. Despite minimal sequence identity, all modeled TM2-Y regions adopt similar domain folds.This conserved architecture likely represents an evolutionarily conserved mechanism for DMV pore formation, Zn-binding interactions facilitate local membrane destabilization preceding pore formation.</p> <h3><strong>Methods</strong></h3> <p>All-atom MD simulations were performed using GROMACS (1) on the NMRbox platform (2) using L40S GPUs. The top-ranked AF3 models for TM2-Y regions from SARS-CoV-2 nsp3 (residues 1499-1945, UniProt P0DTC1), Xinzhou toro-like virus (XTLV) nsp3 (residues 3534-3850, UniProt A0A1L3KIY4) and Simian Hemorrhagic Fever Virus (SHFV) nsp2 (residues 992-1219, UniProt Q68772) were used as starting points.</p> <p>The assembly of explicit lipid bilayer was performed with CHARMM-GUI server (3-5) with lipid ratio 9:1 for POPC:POPG for both membrane leaflets. Initial protein orientation in lipid bilayer was determined using PPM 2.0 server (6). The system was solvated with TIP3P water models (7) and sodium chloride for protein charge compensation. The CHARMM36m force field (8) was applied with periodic boundary conditions in all directions.</p> <p>The initial energy minimization was performed using steepest descent method until convergence. The system was equilibrated over six stages (for 1.875 ns in total) with gradually reduced positional restraints. The temperature was maintained at 303.15 K using velocity rescaling thermostat with coupling time of 1 ps (9), and pressure was maintained at 1 bar using exponential relaxation pressure coupling with time constant of 5 ps. All hydrogen bonds were constrained using the LINCS algorithm (10), and Zn(II) ions were constrained with harmonic restrains to maintain the experimentally observed coordination distances (11).</p> <p>Production simulations (500 ns) were performed with Nose-Hoover thermostat (12). The electrostatic forces were calculated Particle-Mesh Ewald algorithm (13) with cut-off of 1.2 nm. Van der Waals potential was calculated with 1.2 nm cut-off with smoothing to zero function applied after 1.0 nm.</p> <h3><strong>References</strong></h3> <p>1. Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B, Lindahl E.<strong> </strong>2013. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29:845-54.</p> <p>2. Maciejewski MW, Schuyler AD, Gryk MR, Moraru, II, Romero PR, Ulrich EL, Eghbalnia HR, Livny M, Delaglio F, Hoch JC.<strong> </strong>2017. NMRbox: A Resource for Biomolecular NMR Computation. Biophys J 112:1529-1534.</p> <p>3. Wu EL, Cheng X, Jo S, Rui H, Song KC, Davila-Contreras EM, Qi Y, Lee J, Monje-Galvan V, Venable RM, Klauda JB, Im W.<strong> </strong>2014. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J Comput Chem 35:1997-2004.</p> <p>4. Jo S, Kim T, Iyer VG, Im W.<strong> </strong>2008. CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem 29:1859-65.</p> <p>5. Lee J, Cheng X, Swails JM, Yeom MS, Eastman PK, Lemkul JA, Wei S, Buckner J, Jeong JC, Qi Y, Jo S, Pande VS, Case DA, Brooks CL, 3rd, MacKerell AD, Jr., Klauda JB, Im W.<strong> </strong>2016. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J Chem Theory Comput 12:405-13.</p> <p>6. Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL.<strong> </strong>2012. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 40:D370-6.</p> <p>7. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML.<strong> </strong>1983. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics 79:926-935.</p> <p>8. Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, Grubmuller H, MacKerell AD, Jr.<strong> </strong>2017. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71-73.</p> <p>9. Bussi G, Donadio D, Parrinello M.<strong> </strong>2007. Canonical sampling through velocity rescaling. The Journal of chemical physics 126.</p> <p>10. Hess B, Bekker H, Berendsen HJ, Fraaije JG.<strong> </strong>1997. LINCS: a linear constraint solver for molecular simulations. Journal of computational chemistry 18:1463-1472.</p> <p>11. Alberts IL, Nadassy K, Wodak SJ.<strong> </strong>1998. Analysis of zinc binding sites in protein crystal structures. Protein Sci 7:1700-16.</p> <p>12. Evans DJ, Holian BL.<strong> </strong>1985. The nose–hoover thermostat. The Journal of chemical physics 83:4069-4074.</p> <p>13. Darden T, York D, Pedersen L.<strong> </strong>1993. Particle mesh Ewald: An N⋅ log (N) method for Ewald sums in large systems. The Journal of chemical physics 98:10089-10092.</p> |
| format | Recurso digital |
| id | zenodo_https___doi_org_10_5281_zenodo_17653358 |
| institution | Zenodo |
| language | |
| publishDate | 2026 |
| publisher | Zenodo |
| record_format | zenodo |
| spellingShingle | Conserved nsp3 TM2–Y Region in Nidoviruses / MD results Pustovalova, Yulia SARS-CoV-2 Molecular Dynamics Simulation Protein Structure, Tertiary Membrane Proteins Zn-binding protein Arterivirus Tobanivirus Coronavirus <h3><strong>Abstract</strong></h3> <p>Positive-strand RNA viruses remodel host intracellular membranes to form replication organelles in cytoplasm. SARS-CoV-2 and other coronaviruses, which belong to <em>Nidovirales </em>order, replicate within double-membrane vesicles (DMVs) derived from the endoplasmic reticulum that protect newly synthetized viral RNA (vRNA). The vRNA must then be exported to cytoplasm for packaging into virions. A recently discovered SARS-CoV-2 molecular pore complex composed of non-structural proteins 3 and 4 (nsp3 and nsp4) connects the DMV lumen with the cytoplasm, enabling vRNA export. The C-terminal region of nsp3, the Y domain, oligomerizes to form the base of the cytosolic crown of this pore, while preceding transmembrane domain 2 (TM2) anchors the pore into DMV membrane. However, the molecular mechanisms underlying pore formation and whether similar structures are conserved across other <em>Nidovirales </em>members remain unclear. Here, we combined AlphaFold structure prediction, structure-informed multiple sequence alignments, and atomistic molecular dynamics simulations to investigate the conservation and potential function of C-terminal TM2-Y region of nsp3 across nidoviruses. We show that the wedge-shaped TM2, Zn-binding Y1 subdomain, and its adjacent Y2 subdomain constitute a conserved structural module present in most vertebrate-infecting <em>Nidovirales</em> families, including <em>Coronaviridae</em>, <em>Tobaniviridae</em>, <em>Gresnaviridae</em>, <em>Olifoviridae</em> and <em>Arteriviridae</em>. Despite minimal sequence identity, all modeled TM2-Y regions adopt similar domain folds.This conserved architecture likely represents an evolutionarily conserved mechanism for DMV pore formation, Zn-binding interactions facilitate local membrane destabilization preceding pore formation.</p> <h3><strong>Methods</strong></h3> <p>All-atom MD simulations were performed using GROMACS (1) on the NMRbox platform (2) using L40S GPUs. The top-ranked AF3 models for TM2-Y regions from SARS-CoV-2 nsp3 (residues 1499-1945, UniProt P0DTC1), Xinzhou toro-like virus (XTLV) nsp3 (residues 3534-3850, UniProt A0A1L3KIY4) and Simian Hemorrhagic Fever Virus (SHFV) nsp2 (residues 992-1219, UniProt Q68772) were used as starting points.</p> <p>The assembly of explicit lipid bilayer was performed with CHARMM-GUI server (3-5) with lipid ratio 9:1 for POPC:POPG for both membrane leaflets. Initial protein orientation in lipid bilayer was determined using PPM 2.0 server (6). The system was solvated with TIP3P water models (7) and sodium chloride for protein charge compensation. The CHARMM36m force field (8) was applied with periodic boundary conditions in all directions.</p> <p>The initial energy minimization was performed using steepest descent method until convergence. The system was equilibrated over six stages (for 1.875 ns in total) with gradually reduced positional restraints. The temperature was maintained at 303.15 K using velocity rescaling thermostat with coupling time of 1 ps (9), and pressure was maintained at 1 bar using exponential relaxation pressure coupling with time constant of 5 ps. All hydrogen bonds were constrained using the LINCS algorithm (10), and Zn(II) ions were constrained with harmonic restrains to maintain the experimentally observed coordination distances (11).</p> <p>Production simulations (500 ns) were performed with Nose-Hoover thermostat (12). The electrostatic forces were calculated Particle-Mesh Ewald algorithm (13) with cut-off of 1.2 nm. Van der Waals potential was calculated with 1.2 nm cut-off with smoothing to zero function applied after 1.0 nm.</p> <h3><strong>References</strong></h3> <p>1. Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B, Lindahl E.<strong> </strong>2013. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29:845-54.</p> <p>2. Maciejewski MW, Schuyler AD, Gryk MR, Moraru, II, Romero PR, Ulrich EL, Eghbalnia HR, Livny M, Delaglio F, Hoch JC.<strong> </strong>2017. NMRbox: A Resource for Biomolecular NMR Computation. Biophys J 112:1529-1534.</p> <p>3. Wu EL, Cheng X, Jo S, Rui H, Song KC, Davila-Contreras EM, Qi Y, Lee J, Monje-Galvan V, Venable RM, Klauda JB, Im W.<strong> </strong>2014. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J Comput Chem 35:1997-2004.</p> <p>4. Jo S, Kim T, Iyer VG, Im W.<strong> </strong>2008. CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem 29:1859-65.</p> <p>5. Lee J, Cheng X, Swails JM, Yeom MS, Eastman PK, Lemkul JA, Wei S, Buckner J, Jeong JC, Qi Y, Jo S, Pande VS, Case DA, Brooks CL, 3rd, MacKerell AD, Jr., Klauda JB, Im W.<strong> </strong>2016. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J Chem Theory Comput 12:405-13.</p> <p>6. Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL.<strong> </strong>2012. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 40:D370-6.</p> <p>7. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML.<strong> </strong>1983. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics 79:926-935.</p> <p>8. Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, Grubmuller H, MacKerell AD, Jr.<strong> </strong>2017. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71-73.</p> <p>9. Bussi G, Donadio D, Parrinello M.<strong> </strong>2007. Canonical sampling through velocity rescaling. The Journal of chemical physics 126.</p> <p>10. Hess B, Bekker H, Berendsen HJ, Fraaije JG.<strong> </strong>1997. LINCS: a linear constraint solver for molecular simulations. Journal of computational chemistry 18:1463-1472.</p> <p>11. Alberts IL, Nadassy K, Wodak SJ.<strong> </strong>1998. Analysis of zinc binding sites in protein crystal structures. Protein Sci 7:1700-16.</p> <p>12. Evans DJ, Holian BL.<strong> </strong>1985. The nose–hoover thermostat. The Journal of chemical physics 83:4069-4074.</p> <p>13. Darden T, York D, Pedersen L.<strong> </strong>1993. Particle mesh Ewald: An N⋅ log (N) method for Ewald sums in large systems. The Journal of chemical physics 98:10089-10092.</p> |
| title | Conserved nsp3 TM2–Y Region in Nidoviruses / MD results |
| topic | SARS-CoV-2 Molecular Dynamics Simulation Protein Structure, Tertiary Membrane Proteins Zn-binding protein Arterivirus Tobanivirus Coronavirus |
| url | https://doi.org/10.5281/zenodo.17653358 |