Trajectorias de dinamica molecular del zinc finger de las proteinas RAMOSA y SUPERMAN en estado nativo
; Rodrigues, Daniel Enrique ; Garay, Alberto Sergio Otros responsables
Fecha
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
15/05/2024
Fecha de creación:
1/12/2022-1/2/2024
Clasificación temática
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Resumen
Trayectorias de dinámica molecular de la región del ZINC FINGER de las proteínas RAMOSA y SUPERMAN en estado nativo. Usadas para comparar el comportamiento diferencial del mismo en solución.
Métodos
Molecular dynamics studies in solution were carried out using the GROMACS 2021.5 package [31] in conjunction with the AMBER99SB-ILDN force field [32]. The molecular dynamics protocol was performed for a fragment peptide that comprises the zinc fingerdomain regions of SUP (aa: 42–78), which was used in the Nuclear Magnetic Resonance (NMR) structure determination. Indeed, this fragment was chosen from a multiple sequence alignment of plant QALGGH proteins that contains only a single zinc finger domain, most of them from A. thaliana. A multiple sequence alignment of SUP and RA1 proteins identifies the fragment of RA1 (aa: 32–77) as the one corresponding to the peptide from SUP used in the NMR experimental determination. Since experimental structure determination of the RA1 zinc finger region is not available, the initial structure of the RA1 fragment peptide used for starting the molecular dynamics simulation was taken from the AlphaFold best scored model of the complete protein. The AlphaFold server assigns a high level of confidence to the predicted structure of the RA1 zinc finger region. Values of the predicted local distance difference test (pLDDT), which are used to assess the confidence level, are higher than 90/100 for residues from Y46 to H68 and higher than 75/100 for residues from R69 to H83. This initial model has a helix feature spreading beyond the RA1 fragment limits obtained from the multiple sequence alignment; therefore, we decided to use an extended fragment of RA1 (aa: 32–83) for the peptide simulation to avoid disturbing this secondary structure element.
Información Técnica
The simulation protocol was the same for each simulation, and it consisted of total energy minimization of the initial system using the steepest descent energy minimization algorithm and a tolerance of 100 kJ/mol; molecular dynamic simulations with position restraint of the protein heavy atoms for 2 ns in order to allow the water molecules to rearrange around the protein; simulated annealing with position restraint of the zinc finger heavy atoms, increasing the temperature from 300 K to 400 K during 200 ps, equilibrating the temperature for 600 ps, and finally gradually cooling the system back to 300 K during 4.2 ns (5 ns in total) in order to enhance conformational sampling; and molecular dynamics production run at a temperature of 300 K for 1 µs. In all cases, the proteins were inserted into an octahedral box of simple point charge (SPC) water molecules. The minimum distance between the protein and the simulation box was 1.1 nm, to avoid any inappropriate behavior of the water molecules. The timestep of 2 fs was used for the integration of the equations of motions. The Berendsen thermostat and barostat were used to couple the systems to a temperature of 300 K (with coupling time constant 0.1 ps) and a pressure of 1 bar (with coupling time constant 2.0 ps) baths, respectively. The particle mesh Ewald method [35] was used to treat long-range Coulombic interactions. The LINCS algorithm [36] was used to constrain bond lengths of protein atoms and SETTLE for the water molecules. Van der Waals forces were considered up to distances of 1.2 nm, and Coulomb interactions were truncated at 1.2 nm. The root-mean-square deviation (RMSD) of backbone atoms was calculated over the 1µs simulated time, while the root-mean-square fluctuation (RMSF) was computed using the final 200 ns of the molecular dynamic’s trajectory. Both RMSD and RMSF were calculated using the central structure as the reference for rigid fitting and distance calculations.
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