Caracterização biofísica da interação da proteína M2-1 do vírus sincicial respiratório humano (HRSV) com RNA e com os alcaloides esteroidais ciclopamina e solasodina.
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Data
2020-06-25
Autores
Machado, Vitor Brassolatti
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Universidade Estadual Paulista (Unesp)
Resumo
O vírus sincicial respiratório humano (HRSV) é o principal agente causador de doenças crônicas do trato respiratório inferior em crianças e idosos por todo o mundo. A proteína M2-1 do HRSV atua como um fator de antiterminação transcricional, possibilitando a transcrição de todos os genes. Essa proteína é dividida em quatro domínios: um domínio de dedo de zinco; o domínio de oligomerização; o domínio globular que interage com o RNA viral; e um domínio C terminal não estruturado. Em 2016, um estudo publicado por Bailly e colaboradores mostrou que alguns alcaloides esteroidais possuíam atividade antiviral significativa contra o HRSV e que o principal alvo do composto era o domínio globular da proteína M2-1 (dgM2-1). O presente estudo teve como objetivo responder às seguintes perguntas: i) quais são os parâmetros termodinâmicos e as interações não covalentes envolvidas na formação e estabilização do complexo dgM2-1 / RNA ?; ii) ciclopamina e solasodina realmente interagem no domínio globular? Quais regiões destas moléculas estão envolvidas na interação com a proteína? Para alcançar os resultados, mensuramos a supressão de fluorescência do dgM2-1 pelo RNA em diferentes condições de temperatura, força iônica e pH. A constante de dissociação determinada para o complexo é da ordem de 10-9 M e a estequiometria de ligação é de 2 proteínas para 1 molécula de RNA. O efeito da temperatura revelou uma variação negativa na entalpia de ligação, enquanto o aumento no pH ou na concentração salina confirmou que a orientação da interação possui uma componente eletrostática. Os resultados também indicam que a interação é espontânea, com ligações de hidrogênio e forças de van der Waals desempenhando um papel fundamental na estabilização do complexo. Com relação aos alcaloides esteroidais, descobriu-se que ambos interagem no sítio de ligação ao AMP e o complexo formado foi visualizado por meio de docking molecular, o qual demonstrou que a interação ocorre na região de interface interdomínio dgM2-1/dedo de zinco e depende de ligações de hidrogênio e contatos hidrofóbicos para ocorrer. Esses achados necessitam de maiores investigações e são importantes para a compreensão do mecanismo de interação entre a proteína M2-1 do HRSV com o RNA e com candidatos promissores para o desenvolvimento de antivirais.
Human respiratory syncytial virus (HRSV) is the major causative agent of chronic lower respiratory tract diseases in children and elderly worldwide. The M2-1 protein from HRSV acts as a transcription antitermination factor, enabling the transcription of all genes. This protein is divided into four domains: a zinc finger domain; the oligomerization domain; the core domain that interacts with the viral RNA; and unstructured C-terminal domain. In 2016, a study published by Bailly and colleagues showed that steroidal alkaloids had significant antiviral activity against HRSV and that the main target of the compound was the M2-1 protein core domain (cdM2-1). Then, the present study aimed to answer two questions: i) what are the thermodynamic parameters and the non-covalent interactions involved in the formation and stabilization of the cdM2-1/RNA complex? ii) do solasodine and cyclopamine really interact in the core domain? Which regions of these molecules are involved in the interaction with the protein? To achieve the results, we measured the fluorescence quenching of cdM2-1 by RNA under different conditions of temperature, ionic strength and pH. The determined dissociation constant for the complex was on the range of hundreds nanomolar and the binding stoichiometry is of two proteins per RNA molecule. The effect of temperature revealed a negative binding enthalpy change, whereas the increase either in pH or in saline concentration confirmed that electrostatic forces drive the interaction. The results also indicate that it is spontaneous, with hydrogen bonds and van der Waals forces playing a key role on the stabilization of the complex. Regarding to the steroidal alkaloids, it was found that both interact at the AMP binding site and the complex formed was visualized by molecular docking, which demonstrated that the interaction occurs at the cdM2-1 / zinc finger interdomain interface region and it depends on hydrogen bonds and hydrophobic contacts to occur. These findings require further investigation and are important for understanding the mechanism of interaction between HRSV M2-1 protein with RNA and with promising candidates for the development of antivirals.
Human respiratory syncytial virus (HRSV) is the major causative agent of chronic lower respiratory tract diseases in children and elderly worldwide. The M2-1 protein from HRSV acts as a transcription antitermination factor, enabling the transcription of all genes. This protein is divided into four domains: a zinc finger domain; the oligomerization domain; the core domain that interacts with the viral RNA; and unstructured C-terminal domain. In 2016, a study published by Bailly and colleagues showed that steroidal alkaloids had significant antiviral activity against HRSV and that the main target of the compound was the M2-1 protein core domain (cdM2-1). Then, the present study aimed to answer two questions: i) what are the thermodynamic parameters and the non-covalent interactions involved in the formation and stabilization of the cdM2-1/RNA complex? ii) do solasodine and cyclopamine really interact in the core domain? Which regions of these molecules are involved in the interaction with the protein? To achieve the results, we measured the fluorescence quenching of cdM2-1 by RNA under different conditions of temperature, ionic strength and pH. The determined dissociation constant for the complex was on the range of hundreds nanomolar and the binding stoichiometry is of two proteins per RNA molecule. The effect of temperature revealed a negative binding enthalpy change, whereas the increase either in pH or in saline concentration confirmed that electrostatic forces drive the interaction. The results also indicate that it is spontaneous, with hydrogen bonds and van der Waals forces playing a key role on the stabilization of the complex. Regarding to the steroidal alkaloids, it was found that both interact at the AMP binding site and the complex formed was visualized by molecular docking, which demonstrated that the interaction occurs at the cdM2-1 / zinc finger interdomain interface region and it depends on hydrogen bonds and hydrophobic contacts to occur. These findings require further investigation and are important for understanding the mechanism of interaction between HRSV M2-1 protein with RNA and with promising candidates for the development of antivirals.
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Palavras-chave
HRSV, M2-1, RNA, Solasodina, Ciclopamina, Solasodine, Cyclopamine