Conclusões e perspectivas - substituição Molecular

Su 5.4.1 substituição Molecular

6. Conclusões e perspectivas

O domínio catalítico da fator de virulência PlpD de Pseudomonas aeruginosa foi cristalizado na sua forma apo e sua estrutura foi resolvida pela técnica de SAD utilizando-se seleniometionina como espalhador anômalo em combinação com substituição molecular. A resolução máxima do modelo cristalográfico foi de 2.1 Angstroms. As estatísticas obtidas pelo refinamento convergem para um modelo condizente aos dados experimentais, além disso a validação da estrutura pelo gráfico de Ramachandran tornam possíveis a deposição da estrutura no banco de dados PDB. O processo de refinamento encontra-se em fase final de processamento o que indica que os dados cristalográficos ainda poderão ser melhorados, principalmente o B-factor e as porcentagens nas regiões mais favoráveis do grafico de Ramachandran.

A determinação da estrutura cristalográfica da PAL18 de Pseudomonas aeruginosa permitiu uma análise inédita das estruturas e interações presentes no sítio ativo do fator de virulência PlpD que é expresso em linhagens que não codificam para ExoU. Comparações estruturais com o outro membro da família reveleu diferenças interessantes na região do sítio ativo. Principalmente a presença do resíduo de aspartato que compõe a díade catalítica na estrutura, o que permite o uso de simulações de modelagem molecular e docking, técnicas que podem auxiliar na determinação da formação do complexo enzima-substrato.

Outras diferenças como a posição do resíduo responsável por atuar como oxiânion, fundamental na atividade enzimática, revelam uma provável diferença entre o modo de interação com o substrato das proteínas PlpD e ExoU. Através da comparação lipases humana e bacteriana sugere-se que a região flexível presente no modelo da PAL18 forme a tampa conservada nessa família de enzimas. Essa tampa provavelmente cumpra um papel essencial na proteção contra autocatálise de lipídeos da própria P. aeruginosa, uma vez que essa enzima não necessite de co-fatores eucarióticos para ativação.

Esse projeto iniciou-se a partir de um convite da Dra Sophie Bleves, do laboratório de engenharia de sistemas macromoleculares em Marselha, França. O grupo da Dra identificou e caracterizou inicialmente a proteína PlpD, a determinação estrutural do domínio catalítico dessa proteína juntamente com as investigações biológicas realizadas pelo grupo em Marselha, tem por objetivo a busca de alvos farmacológicos que possam inibir a atividade da

enzima importante para o processo infeccioso do patógeno. Além do fator de virulência secretado PAL18 os demais componentes da proteína PlpD também estão sendo investigados pelo grupo da Dra Bleves de modo a fornecer um entendimento global da proteína e que juntamente com a estrutura da PAL18 será compilado em um artigo.

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8. Anexos

No documento Determinação da estrutura tridimensional do domínio catalítico do fator de virulência PlpD de Pseudomonas aeruginosa (páginas 76-86)