Análisis de las interacciones entre las proteínas Transcriptasa inversa (RT) y DNA del virus de la inmunodeficiencia humana tipo 1 (VIH-1) a través de dinámica molecular.
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Fecha
2023
Autores
Pertuz Peña, María Fernanda
Puello Guerra, Natalia Melitza
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
El VIH ha sido un tema de preocupación a nivel global y continúa siendo uno de los desafíos más apremiantes en la esfera de la salud pública a nivel mundial. La elevada diversidad genética del virus ha dado lugar a mutaciones en la RT que confieren resistencia a diversos inhibidores de la RT. Por lo tanto, esta tesis se centró en analizar las interacciones entre las proteínas RT y el ADN del virus de la Inmunodeficiencia Humana tipo 1 (VIH-1) mediante dinámica molecular. El estudio comenzó con la búsqueda de secuencias en la base de datos Protein Data Bank (PDB) que contienen cristales de los complejos RT WT-DNA y RT mutante-DNA. Para obtener estructuras iniciales, se utilizó un modelo generado por AlphaFold2 para la RT mutante, debido a que el cristal encontrado tenía Gaps, mientras que se usó la estructura del PDB para la RT WT. A su vez, se llevó a cabo un modelado de la proteína mutante, seleccionando el mejor candidato generado por AlphaFold2 y validando su estructura con la herramienta MatchMaker. Posteriormente, se preparó el sistema para la simulación de dinámica molecular, que implicó la adición de iones y agua, así como la parametrización de las interacciones entre átomos y moléculas utilizando campos de fuerza específicos. Simultáneamente, se realizó una fase de minimización en el sistema, seguida de calentamiento y equilibrio bajo condiciones de presión constante. Finalmente, se llevó a cabo una simulación de producción de 100 ns.
La relevancia biológica de estas mutaciones radica en su potencial impacto en la eficacia de los tratamientos antirretrovirales y la capacidad del VIH-1 para resistirlos. Es por esta razón que resultados revelaron diferencias significativas en la estructura y flexibilidad entre la RT mutante y la RT WT, lo que sugiere que las mutaciones pueden influir en la estabilidad y la función de la proteína.
Además, se calculó la energía libre de unión a través de mecánica molecular - área de superficie basada en Poisson-Boltzmann (MM/PBSA) para evaluar la afinidad entre la RT y el ADN. Posteriormente, se identificaron residuos específicos que contribuyen
significativamente a la estabilidad del complejo. Asimismo, se analizaron las interacciones entre residuos a lo largo de la simulación, identificando cinco categorías de interacciones: aniónicas, hidrofóbicas, contactos de van der Waals, interacciones tipo pi-stacking y enlaces donadores y aceptores de puentes de hidrógeno. Los resultados revelaron diferencias en las interacciones en la RT mutante y la RT WT, lo que proporciona información valiosa sobre cómo las mutaciones pueden alterar la dinámica molecular de la proteína.
Los resultados sugieren la hipótesis de que la RT mutante, al tener un mayor número de interacciones en los tipos de Anionic, VdWcontact y HBAaceptor, podría estar mejor preparada para resistir la acción de los fármacos antirretrovirales. Por otro lado, la RT WT, al carecer de estas interacciones en la misma medida, podría ser menos eficaz en la resistencia a los medicamentos.
Para concluir, esta tesis utilizó la dinámica molecular para analizar las interacciones entre las proteínas RT y el ADN del VIH-1, proporcionando información esencial sobre cómo las mutaciones en la RT pueden afectar su estructura y función, así como su capacidad de resistir a los tratamientos antirretrovirales. Los resultados obtenidos tienen implicaciones significativas en la investigación y el tratamiento del VIH-1.
HIV has been an issue of global concern and continues to be one of the most pressing public health challenges worldwide. The high genetic diversity of the virus has resulted in mutations in RT that confer resistance to various RT inhibitors. Therefore, this thesis focused on analyzing the interactions between RT proteins and Human Immunodeficiency Virus type 1 (HIV-1) DNA by molecular dynamics. The study began by searching the Protein Data Bank (PDB) database for sequences containing crystals of the RT WT-DNA and RT mutant-DNA complexes. To obtain initial structures, a model generated by AlphaFold2 was used for mutant RT, because the crystal found had Gaps, while the PDB structure was used for WT RT. In turn, a modeling of the mutant protein was carried out, selecting the best candidate generated by AlphaFold2 and validating its structure with the MatchMaker tool. Subsequently, the system was prepared for molecular dynamics simulation, which involved the addition of ions and water, as well as the parameterization of interactions between atoms and molecules using specific force fields. Simultaneously, a minimization phase was performed on the system, followed by heating and equilibrium under constant pressure conditions. Finally, a 100 ns production simulation was carried out. The biological relevance of these mutations lies in their potential impact on the efficacy of antiretroviral treatments and the ability of HIV-1 to resist them. It is for this reason that results revealed significant differences in structure and flexibility between mutant RT and WT RT, suggesting that mutations may influence protein stability and function. In addition, binding free energy was calculated via molecular mechanics-Poisson-Boltzmann-based surface area (MM/PBSA) to assess the affinity between RT and DNA. Subsequently, specific residues that contribute significantly to the stability of the complex were identified. Furthermore, the interactions between residues throughout the simulation were analyzed, identifying five categories of interactions: anionic, hydrophobic, van der Waals contacts, pi-stacking interactions, and hydrogen bridge donor and acceptor bonds. The results revealed differences in the interactions in mutant RT and WT RT, providing valuable information on how mutations can alter the molecular dynamics of the protein. The results suggest the hypothesis that mutant RT, having a greater number of interactions in the Anionic, VdWcontact and HBAaceptor types, might be better able to resist the action of antiretroviral drugs. On the other hand, RT WT, lacking these interactions to the same extent, might be less effective in drug resistance. To conclude, this thesis used molecular dynamics to analyze the interactions between RT proteins and HIV-1 DNA, providing essential information on how mutations in RT may affect its structure and function, as well as its ability to resist antiretroviral treatments. The results obtained have significant implications for HIV-1 research and treatment.
HIV has been an issue of global concern and continues to be one of the most pressing public health challenges worldwide. The high genetic diversity of the virus has resulted in mutations in RT that confer resistance to various RT inhibitors. Therefore, this thesis focused on analyzing the interactions between RT proteins and Human Immunodeficiency Virus type 1 (HIV-1) DNA by molecular dynamics. The study began by searching the Protein Data Bank (PDB) database for sequences containing crystals of the RT WT-DNA and RT mutant-DNA complexes. To obtain initial structures, a model generated by AlphaFold2 was used for mutant RT, because the crystal found had Gaps, while the PDB structure was used for WT RT. In turn, a modeling of the mutant protein was carried out, selecting the best candidate generated by AlphaFold2 and validating its structure with the MatchMaker tool. Subsequently, the system was prepared for molecular dynamics simulation, which involved the addition of ions and water, as well as the parameterization of interactions between atoms and molecules using specific force fields. Simultaneously, a minimization phase was performed on the system, followed by heating and equilibrium under constant pressure conditions. Finally, a 100 ns production simulation was carried out. The biological relevance of these mutations lies in their potential impact on the efficacy of antiretroviral treatments and the ability of HIV-1 to resist them. It is for this reason that results revealed significant differences in structure and flexibility between mutant RT and WT RT, suggesting that mutations may influence protein stability and function. In addition, binding free energy was calculated via molecular mechanics-Poisson-Boltzmann-based surface area (MM/PBSA) to assess the affinity between RT and DNA. Subsequently, specific residues that contribute significantly to the stability of the complex were identified. Furthermore, the interactions between residues throughout the simulation were analyzed, identifying five categories of interactions: anionic, hydrophobic, van der Waals contacts, pi-stacking interactions, and hydrogen bridge donor and acceptor bonds. The results revealed differences in the interactions in mutant RT and WT RT, providing valuable information on how mutations can alter the molecular dynamics of the protein. The results suggest the hypothesis that mutant RT, having a greater number of interactions in the Anionic, VdWcontact and HBAaceptor types, might be better able to resist the action of antiretroviral drugs. On the other hand, RT WT, lacking these interactions to the same extent, might be less effective in drug resistance. To conclude, this thesis used molecular dynamics to analyze the interactions between RT proteins and HIV-1 DNA, providing essential information on how mutations in RT may affect its structure and function, as well as its ability to resist antiretroviral treatments. The results obtained have significant implications for HIV-1 research and treatment.
Descripción
Palabras clave
VIH-1, Retrovirus, Bioinformática, DNA, Transcriptasa inversa, Dinámica molecular, HIV-1, Retrovirus, Bioinformatics, DNA, Reverse transcriptase, Molecular dynamics