Estudos estruturais com a importina: Agnes Alessandra Sekijima Takeda. -
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2009-02-11
Autores
Takeda, Agnes Alessandra Sekijima [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A Importina-α (ImpA) participa da via clássica de importação nuclear reconhecendo seqüências de localização nuclear (NLS) presentes em proteínas que apresentam atividades no núcleo. Almejando mais informações a respeito do mecanismo de reconhecimento para importação nuclear, nesse trabalho foram efetuados experimentos de expressão, purificação e cristalografia de raios-X da ImpA de Mus musculus com o peptídeo NLS da proteína de reparo de DNA Ku70 e peptídeos TMNLS, T52NLS, frutos de bibliotecas de peptídeos NLS específicos para isoformas da ImpA de Mus musculus e Homo sapiens, respectivamente. O peptídeo TMNLS, conforme esperado, ligou-se à ImpA de maneira similar ao NLS do antígeno T da SV40. Já o peptídeo não clássico T52NLS, obtido de uma biblioteca de peptídeos NLS para a isoforma 5 de Homo sapiens, acomodou-se ao sítio principal da ImpA de maneira satisfatória, indicando que essa seqüência também apresenta afinidade à isoforma de Mus musculus. Complementar à esse resultado, foi constatada a ligação do T52NLS ao sítio secundário da ImpA e, pela primeira vez, foi observada a ligação de um peptídeo monopartido de maneira alternativa, paralela à ImpA, ao contrário da posição anti-paralela, convencional. Surpreendentemente, resultados do complexo ImpA-NLS de Ku70 indicaramno como monopartido, e ligando-se a ImpA de maneira similar à versão fosforilada do peptídeo NLS do antígeno T da SV40. Posições específicas nos sítios de ligação foram confirmadas como essenciais, bem como resíduos da ImpA conservados nessas regiões, indicando a importância das interações intermoleculares nesses sítios. Adicionalmente foram obtidas informações relevantes, como a importância de resíduos de prolina nos NLSs e a não obrigatoriedade de regiões altamente básicas para o reconhecimento de um NLS pela ImpA
The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS
The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS
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Palavras-chave
Biologia molecular, Bioquímica, Nuclear import
Como citar
TAKEDA, Agnes Alessandra Sekijima. Estudos estruturais com a importina: Agnes Alessandra Sekijima Takeda. -. 2009. 89 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2009.