Estudos moleculares com a bothropstoxina-I, uma miotoxina de Bothrops jararacussu: estudo dinâmico do seu espaço conformacional
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Data
2019-08-01
Autores
Gomes, Antoniel Augusto Severo
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Acidentes ofídicos são considerados um problema de saúde pública global, dada sua alta ocorrência de mortes, onde alguns casos resultam em sequelas irreparáveis, deixando o indivíduo desabilitado por toda a vida. Somente no Brasil, em média 20 mil mortes ao ano são registradas. Desta forma, alguns pesquisadores têm buscado estudar a estrutura-função de componentes dos venenos de serpentes peçonhentas à fim de desenvolver alternativas terapêuticas para esse problema de grande interesse médico. Um dos componentes que tem recebido atenção nas últimas décadas é a BthTX-I, uma proteína PLA2s-homóloga não catalítica, devido seu efeitos farmacológico notadamente miotóxico. Embora diversas técnicas tenham sido empregadas na compreensão do mecanismo de ação dessas proteínas, o conhecimento nessa área permanece controverso, tornando-se necessário a continuidade de estudos. O estudo estrutural e computacional de proteínas constitui uma alternativa viável para identificar as várias conformações de uma dada proteína, bem como seu comportamento dinâmico. Sendo assim, o presente trabalho buscou aplicar métodos computacionais, como simulação de MD, análise de Modos Normais (NM) e Dinâmica Molecular excitada por Modos Normais (MDeNM) à BthTX-I em pH ácido ou básico e nativa ou em complexo com o α-tocoferol – molécula tida como ativadora alostérica – na descrição do seu espaço conformacional, buscando compreender os aspectos estruturais do seu mecanismo de ação. Além disso, foram empregadas técnicas em solução, como Espalhamento de Luz Dinâmico (DLS), a fim de compreender o estado oligomérico da proteína frente ao α-tocoferol. Os resultados de MD mostraram que a BthTX-I possui estabilidade dimérica em pH neutro/básico. Já os experimentos de MDeNM mostraram que esta técnica é mais eficiente que a MD para descrever o espaço conformacional da BthTX-I, especialmente quando movimentos de alta amplitude são desejados, graças à sua combinação com os modos normais, causando altas alterações conformacionais do sítio de Ruptura de Membrana (MDiS), sendo exposto para o solvente e se aproximando do plano de sulfato que mimetiza a membrana celular. Tal movimento é altamente relacionado com a descrição dos ângulos de Euler, sugerindo que a exposição do MDiS é resultado de alterações da estrutura quaternária da BthTX-I. A liberdade do MDiS pode ser explicada pela perturbação da sua interação com a Hélice-I da BthTX-I causada pela presença do α-tocoferol, definindo assim uma característica importante para um ativador alostérico. Por fim, os experimentos de DLS reforçaram o caráter oligomerizante do α-tocoferol frente a BthTX-I, uma característica chave para a miotoxicidade dessa classe de proteínas.
Ophidian accidents constitute a global public health problem due its high mortality, with some cases leading to permanent sequels. In Brazil, around 20 thousand deaths are registered. Therefore, researchers have been studying the snake venom composition in order to develop therapeutic alternatives to this medical problem. Some components from snake venom are highlighted in the past decades, as BthTX-I, a non-catalytic PLA2s-homologous protein, due its many pharmacological effects, notably myotoxic. Although many techniques have been employed to better understand the molecular mechanism of action for these proteins, this knowledge remains controversial, emphasizing the need to progress this field. The structural and computational study of proteins is a valuable tool to access the various conformations of a given protein, as well its dynamical behavior. Thus, the present work applied computational methods as MD, Normal Modes (NM) analysis and Molecular Dynamics with excited Normal Modes (MDeNM) to BthTX-I in acidic or basic pH and unbound or bound to α-tocopherol – a molecule identified as an allosteric activator – in order to describe the conformational space of this protein and determine the structural aspects of its mechanism of action. Further, biophysical experiments as Dynamic Light Scattering (DLS) were employed in order to comprehend the oligomeric form of BthTX-I with or without α-tocopherol. MD simulations showed that dimeric form of BthTX-I is stable when in neutral/basic pH conditions. MDeNM showed as a better tool to describe the conformational space of BthTX-I, specially when high amplitude movements are desired, due its mixing with normal modes, leading to high conformational changes of the Membrane-Disruptin site (MDiS), which were exposed to the solvent and approximated to the sulphate plane that mimics the cell membrane surface. This movement is highly related with the Euler angle description, suggesting that the MDiS exposure is result of quaternary movements of BthTX-I. The liberty of MDiS might be explained due the perturbation of its interaction with Helix-I of the protein caused by the presence α-tocopherol, describing an important feature that defines an allosteric activator. Furthermore, DLS experiments reinforced the ability of α-tocopherol to induce the BthTX-I oligomerization, a key property for myotoxicity for this class of proteins.
Ophidian accidents constitute a global public health problem due its high mortality, with some cases leading to permanent sequels. In Brazil, around 20 thousand deaths are registered. Therefore, researchers have been studying the snake venom composition in order to develop therapeutic alternatives to this medical problem. Some components from snake venom are highlighted in the past decades, as BthTX-I, a non-catalytic PLA2s-homologous protein, due its many pharmacological effects, notably myotoxic. Although many techniques have been employed to better understand the molecular mechanism of action for these proteins, this knowledge remains controversial, emphasizing the need to progress this field. The structural and computational study of proteins is a valuable tool to access the various conformations of a given protein, as well its dynamical behavior. Thus, the present work applied computational methods as MD, Normal Modes (NM) analysis and Molecular Dynamics with excited Normal Modes (MDeNM) to BthTX-I in acidic or basic pH and unbound or bound to α-tocopherol – a molecule identified as an allosteric activator – in order to describe the conformational space of this protein and determine the structural aspects of its mechanism of action. Further, biophysical experiments as Dynamic Light Scattering (DLS) were employed in order to comprehend the oligomeric form of BthTX-I with or without α-tocopherol. MD simulations showed that dimeric form of BthTX-I is stable when in neutral/basic pH conditions. MDeNM showed as a better tool to describe the conformational space of BthTX-I, specially when high amplitude movements are desired, due its mixing with normal modes, leading to high conformational changes of the Membrane-Disruptin site (MDiS), which were exposed to the solvent and approximated to the sulphate plane that mimics the cell membrane surface. This movement is highly related with the Euler angle description, suggesting that the MDiS exposure is result of quaternary movements of BthTX-I. The liberty of MDiS might be explained due the perturbation of its interaction with Helix-I of the protein caused by the presence α-tocopherol, describing an important feature that defines an allosteric activator. Furthermore, DLS experiments reinforced the ability of α-tocopherol to induce the BthTX-I oligomerization, a key property for myotoxicity for this class of proteins.
Descrição
Palavras-chave
Bothropstoxina-I, MD, Modos normais, PLA2s-homólogas, Veneno de serpente, Normal modes, PLA2s-homologous, Snake venom