Caracterização biofísica da interação entre a proteína M2-1 do Vírus Sincicial Respiratório Humano (HRSV) com quercetina
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Data
2016-02-25
Autores
Teixeira, Thiago Salem Pançonato [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O Vírus Sincicial Respiratório Humano (hRSV) é o principal agente causador de doenças respiratórias agudas severas, como pneumonia e bronquiolite. Trata-se de um vírus da família Paramyxoviridae, com simetria helicoidal e envelope lipídico. Possui um genoma de RNA fita simples, não segmentado, com 10 genes codificantes. Um dos fatores que contribuem para o sucesso na replicação viral é a interação da proteína M2-1 com as proteínas P, M e com o RNA. Tal proteína age através da interação com RNA, e assim, participa efetivamente no processo de replicação viral, apresentando atividade anti-parada da replicação sendo ativa quando na forma tetramérica. Deste modo, o ligante que realizar interação com a M2-1 é um potencial candidato a prevenir as interações M2-1 com RNA e demais proteínas, impedindo deste modo o sucesso da replicação viral. Flovonoides são compostos naturais com várias atividades como antioxidante, anticancerígena, cardio protetora, antibacteriana e antiviral. Quercetina é um flavonoide que apresenta atividade antiviral, inibindo o complexo de replicação. Com a finalidade de investigar a interação de um potencial candidato a inibição da atividade da M2-1, clonamos, expressamos e purificamos a proteína M2-1 do hRSV e, determinamos sua característica físico-química de interação com quercetina em diferentes condições de temperatura. Para clonagem e expressão do gene M2-1 foram escolhidos o vetor pGEX-4T-1 e a bactéria Escherichia coli da linhagem BL21 Gold (DE3). A proteína expressa foi purificada em cromatografia de afinidade e cromatografia de exclusão molecular. As análises de composição de estrutura secundária e estabilidade térmica foram realizadas por espectroscopia de dicroísmo circular e, a interação com Quercetina com espectroscopia de fluorescência. Para o mapeamento da interação com a Quercetina foram realizadas docking molecular e dinâmica molecular. A proteína M2-1 purificada apresenta 40% α-hélice, 10% folhas β e 19% alças e 31% random coils, sendo a temperatura de melting de 55,6°C. O teste de titulação com o flavonoide Quercetina mostrou que ela interage em dois pontos, no sítio 1 (constante de afinidade ~1,5x106 M-1) estabelece uma interação do tipo iônica e no sítio 2 (1,1x105 M-1) estabelece uma interação do tipo hidrofóbica. Uma molécula de Quercetina interage no sítio 1 (formada pelo tetrâmero) e quatro moléculas interagem no sítio 2 (uma por unidade proteica). O estudo de dinâmica molecular mostra que o domínio central (composto de α-hélice) é estável e, a extremidade N-terminal adquire maior estabilidade quando a proteína está na forma tetramérica. O docking do ligante Quercetina mostra que ela pode interagir na face N-terminal com interação do tipo iônica e entre cadeias próxima aos domínios de oligomerização e ligação ao RNA e proteína P, com tipo de interação hidrofóbica. Os dados teóricos reforçam os dados experimentais, mostrando que a quercetina interage com a proteína M2-1, principalmente no poro central da proteína.
Human Respiratory Syncytial Virus (hRSV) is the major causative agent of acute respiratory infections such as pneumonia and bronchiolitis. This is a virus of the Paramyxoviridae family with helical symmetry and lipid envelope. It has a single strand RNA genome, not segmented, with 10 coding genes. One of the factors that contribute to success in viral replication is the interaction of M2-1 protein with P, M protein and RNA viral. Such protein acts through the interaction with RNA, hence, participating in the viral replication process, having anti-stop activity in the replication process and, the protein is active only as a tetramer. Thus, a ligand that interacts with M2-1 is a potential candidate to prevent interaction of M2-1 with other proteins, thereby preventing the success of viral replication. The aim of the research is to clone, express and purify the M2-1 protein of hRSV and then determine its physicochemical characteristics of the interaction with Quercetin in different temperatures to investigate the interaction of a potential candidate to inhibit the activity of the M2-1. The M2-1 gene was cloned in vector pGEX-4T-1 and expressed in Escherichia coli strain BL21 Gold (DE3). The expressed protein was purified on affinity chromatography molecular exclusion chromatography, thermal stability and secondary structure were analyzed by circular dicroism and the interaction with Quercetin was analyzed by fluorescence spectroscopy. To understand the interaction mechanism was performed molecular dynamics and molecular docking. The purified protein has 40% α-helix, 10% β-sheet, 19% turns and 31% random coils, being the melting temperature of 55,6°C. The titration of M2- 1 with Quercetin showed that it interacts in two points, on site 1 (the binding affinity is ~1,5x106 M -1 ) and stablishes an ionic interaction and, on site 2 (1,1x105 M -1 ) stablishes a hydrophobic interaction. One Quercetin molecule interacts on site 1 (formed by the tetramer) and four Quercetins interacts on site 2 (one per protein unity). The molecular dynamics study shows that the central domain (composed mainly by α-helix) is stable and, the N-terminal extremity gets more stabilization when the protein is in the tetrameric form. The molecular docking of Quercetin shows that it can interact on N-terminus face with ionic interaction pattern and between chains close to oligomerization and RNA and P protein interaction domains with a hydrophobic interaction pattern. Hence, this ligand could be inhibiting the protein activity by disrupting it.
Human Respiratory Syncytial Virus (hRSV) is the major causative agent of acute respiratory infections such as pneumonia and bronchiolitis. This is a virus of the Paramyxoviridae family with helical symmetry and lipid envelope. It has a single strand RNA genome, not segmented, with 10 coding genes. One of the factors that contribute to success in viral replication is the interaction of M2-1 protein with P, M protein and RNA viral. Such protein acts through the interaction with RNA, hence, participating in the viral replication process, having anti-stop activity in the replication process and, the protein is active only as a tetramer. Thus, a ligand that interacts with M2-1 is a potential candidate to prevent interaction of M2-1 with other proteins, thereby preventing the success of viral replication. The aim of the research is to clone, express and purify the M2-1 protein of hRSV and then determine its physicochemical characteristics of the interaction with Quercetin in different temperatures to investigate the interaction of a potential candidate to inhibit the activity of the M2-1. The M2-1 gene was cloned in vector pGEX-4T-1 and expressed in Escherichia coli strain BL21 Gold (DE3). The expressed protein was purified on affinity chromatography molecular exclusion chromatography, thermal stability and secondary structure were analyzed by circular dicroism and the interaction with Quercetin was analyzed by fluorescence spectroscopy. To understand the interaction mechanism was performed molecular dynamics and molecular docking. The purified protein has 40% α-helix, 10% β-sheet, 19% turns and 31% random coils, being the melting temperature of 55,6°C. The titration of M2- 1 with Quercetin showed that it interacts in two points, on site 1 (the binding affinity is ~1,5x106 M -1 ) and stablishes an ionic interaction and, on site 2 (1,1x105 M -1 ) stablishes a hydrophobic interaction. One Quercetin molecule interacts on site 1 (formed by the tetramer) and four Quercetins interacts on site 2 (one per protein unity). The molecular dynamics study shows that the central domain (composed mainly by α-helix) is stable and, the N-terminal extremity gets more stabilization when the protein is in the tetrameric form. The molecular docking of Quercetin shows that it can interact on N-terminus face with ionic interaction pattern and between chains close to oligomerization and RNA and P protein interaction domains with a hydrophobic interaction pattern. Hence, this ligand could be inhibiting the protein activity by disrupting it.
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Palavras-chave
HRSV, M2-1, Quercetina, Dinâmica molecular, Quercetin, Molecular dynamics