Structural studies with importin-α from Neurospora crassa and nuclear localization sequences (NLS) of proteins related with fungi metabolism

Author(s)

Natália Elisa Bernardes

• Document type: Doctoral Thesis
• Press: Botucatu. 2018-08-15.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2018-07-06
• Advisor: Marcos Roberto de Mattos Fontes

Abstract

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. (AU)

• FAPESP's process: 14/10289-0 - Structural studies with importin-± and nuclear localization sequences (NLS) of proteins related with fungi metabolism
• Grantee: Natália Elisa Bernardes
• Support Opportunities: Scholarships in Brazil - Doctorate