Estudos estruturais com a importina-α do fungo Neurospora crassa e peptídeos de sequências de localização nuclear (NLS) de proteínas relacionadas ao metabolismo de fungos
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Data
2018-07-06
Autores
Bernardes, Natália Elisa [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A comunicação entre o núcleo celular e o citoplasma acontece através de mecanismos de transporte que permitem a passagem de moléculas por poros presentes no envoltório nuclear. Na Via Clássica de Importação Nuclear, a proteína Importina-α (Impα) atua na identificação das proteínas a serem transportadas ao núcleo a partir do reconhecimento de sequências de localização nuclear (NLS). Os primeiros estudos de caracterização estrutural da Impα de Neurospora crassa (NcImpα) e a sua estrutura cristalográfica mostraram a presença de regiões que podem estar relacionadas a especificidades da proteína NcImpα no reconhecimento de NLSs de proteínas de fungos. Além disso, foram reconhecidos prováveis NLSs em proteínas reguladoras do metabolismo de carbono e nitrogênio em fungos. O objetivo desse trabalho é identificar e caracterizar o modo de interação de NLSs de proteínas fúngicas com a NcImpα e verificar possíveis especificidades da proteína NcImpα no reconhecimento de NLSs. Os potenciais peptídeos NLS de proteínas dos fungos filamentosos N. crassa e Aspergillus nidulans foram submetidos a experimentos de calorimetria de titulação isotérmica (ITC) com a proteína NcImpα, para calcular a afinidade entre as moléculas. Dentre os peptídeos testados, as sequências correspondentes ao potenciais NLS dos fatores de transcrição PAC-3, NIT-2, FLB-3, VOSA e VEA, apresentaram elevada afinidade com a NcImpα, conforme indicado pelos valores de Kd obtidos. Quando submetidos a experimentos de importação nuclear,o peptídeo NIT2-NLS foi eficientemente transportado para o interior do núcleo celular. Foram realizados testes de cristalização para a elucidação da estrutura dos complexos Impα/peptídeos NLS. O conjunto de dados do complexo NcImpα/NIT2NLS a 2,50 Å de resolução, permitiu a elucidação da estrutura onde foi possível observar a presença do peptídeo NIT2-NLS nos sítios principal e secundário de NcImpα. Já a estrutura do complexo MmImpα/PAC3-NLS mostrou o peptídeo se lihando com maior afinidade apenas ao sítio prinicpal. A comparação das sequências dos peptídeos VEA-NLS e VE1-NLS mostrou que a substituição de um resíduo de lisina por uma leucina e uma lisina por uma arginina nas posições P1’ e P2 é suficiente para reduzir drasticamente a afinidade do peptídeo VE1-NLS pela NcImpα. Por fim, se sugere que o peptídeo VOSA-NLS se liga a NcImpα em uma conformação muito semelhante ao peptídeo NIT2-NLS e as NLSs da proteína FLB3-NLS podem apresentam afinidade a apenas um dos sítios da proteína NcImpα.
The communication between the cell nucleus and the cytoplasm happens through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. In the classical nuclear import pathway, the protein Importin-α (Impα) acts in the identification of the proteins to be transported to the nucleus from the recognition of nuclear localization sequences (NLS). The first structural characterization studies of N. crassa Impα (NcImpα) and its crystallographic structure showed the presence of regions that may be related to protein specificities in the recognition of fungal NLSs. In addition, NLSs were recognized in proteins related to fungal metabolism. The objective of this work is to identify NLSs in fungal proteins and observe the specificities of the NcImpα protein. Potential NLS peptides of N. crassa were subjected to isothermal titration calorimetry (ITC) experiments with NcImpα to verify and calculate the affinity of the complexes. Among the peptides tested, the sequences corresponding to the potentials NLS of the transcription factors PAC-3, NIT-2, FLB-3, VOSA and VEA, showed high affinity with NcImpα, as indicated by the Kd values obtained. When subjected to functional experiments with HeLa cells, the peptide NIT2-NLS were efficiently transported into the cell nucleus. Crystallization tests were performed to elucidate the structure of the complexes Impα/NLS peptides. A set of data from the NcImpα/ NIT2NLS complex was collected, with 99.71% completeness at 2.50 resolution, from which the structure was elucidated. It was possible to observe the presence of NIT2NLS peptides in the major and minor binding sites of NcImpα. The structure of the MmImpα/PAC3-NLS complex was also elucidated in order to compare the interaction of the same peptide with different isoforms of the Impα protein. The structure of the MmImpα/PAC3-NLS complex showed the peptide binding with higher affinity to the major binding site. Comparison of the sequences of the VEA-NLS and VE1-NLS peptides showed that the substitution of a lysine residue by a leucine and a lysine by an arginine at positions P1 'and P2 is sufficient to drastically reduce the affinity of the peptide VE1-NLS by NcImpα. Finally, it is suggested that the VOSA-NLS peptide binds to NcImpα in a conformation very similar to the peptide NIT2-NLS and the NLSs of the FLB3-NLS protein exhibits affinity to only one of the NcImpα protein sites.
The communication between the cell nucleus and the cytoplasm happens through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. In the classical nuclear import pathway, the protein Importin-α (Impα) acts in the identification of the proteins to be transported to the nucleus from the recognition of nuclear localization sequences (NLS). The first structural characterization studies of N. crassa Impα (NcImpα) and its crystallographic structure showed the presence of regions that may be related to protein specificities in the recognition of fungal NLSs. In addition, NLSs were recognized in proteins related to fungal metabolism. The objective of this work is to identify NLSs in fungal proteins and observe the specificities of the NcImpα protein. Potential NLS peptides of N. crassa were subjected to isothermal titration calorimetry (ITC) experiments with NcImpα to verify and calculate the affinity of the complexes. Among the peptides tested, the sequences corresponding to the potentials NLS of the transcription factors PAC-3, NIT-2, FLB-3, VOSA and VEA, showed high affinity with NcImpα, as indicated by the Kd values obtained. When subjected to functional experiments with HeLa cells, the peptide NIT2-NLS were efficiently transported into the cell nucleus. Crystallization tests were performed to elucidate the structure of the complexes Impα/NLS peptides. A set of data from the NcImpα/ NIT2NLS complex was collected, with 99.71% completeness at 2.50 resolution, from which the structure was elucidated. It was possible to observe the presence of NIT2NLS peptides in the major and minor binding sites of NcImpα. The structure of the MmImpα/PAC3-NLS complex was also elucidated in order to compare the interaction of the same peptide with different isoforms of the Impα protein. The structure of the MmImpα/PAC3-NLS complex showed the peptide binding with higher affinity to the major binding site. Comparison of the sequences of the VEA-NLS and VE1-NLS peptides showed that the substitution of a lysine residue by a leucine and a lysine by an arginine at positions P1 'and P2 is sufficient to drastically reduce the affinity of the peptide VE1-NLS by NcImpα. Finally, it is suggested that the VOSA-NLS peptide binds to NcImpα in a conformation very similar to the peptide NIT2-NLS and the NLSs of the FLB3-NLS protein exhibits affinity to only one of the NcImpα protein sites.
Descrição
Palavras-chave
Cristalografia, NLS, ITC, Neurospora, Crystallography, NIT2