Comparative study of the interactions between fungal transcription factor nuclear localization sequences with mammalian and fungal importin-alpha

Natália E Bernardes,Marcos R M Fontes,Thiago R Dreyer,Maria Célia Bertolini,Cintia A Fukuda,Hamine C De Oliveira,Tainá D Da Silva,Andrea C De Barros

doi:10.1038/s41598-020-58316-9

Natália E Bernardes, Marcos R M Fontes + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-58316-9

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Journal: Scientific Reports	Publication Date: Jan 29, 2020
Citations: 8	License type: open-access

Affiliation: Universidade Estadual Paulista (Unesp)

Abstract

Importin-α (Impα) is an adaptor protein that binds to cargo proteins (containing Nuclear Localization Sequences - NLSs), for their translocation to the nucleus. The specificities of the Impα/NLS interactions have been studied, since these features could be used as important tools to find potential NLSs in nuclear proteins or even for the development of targets to inhibit nuclear import or to design peptides for drug delivery. Few structural studies have compared different Impα variants from the same organism or Impα of different organisms. Previously, we investigated nuclear transport of transcription factors with Neurospora crassa Impα (NcImpα). Herein, NIT-2 and PAC-3 transcription factors NLSs were studied in complex with Mus musculus Impα (MmImpα). Calorimetric assays demonstrated that the PAC-3 NLS peptide interacts with both Impα proteins with approximately the same affinity. The NIT-2 NLS sequence binds with high affinity to the Impα major binding site from both organisms, but its binding to minor binding sites reveals interesting differences due to the presence of additional interactions of NIT-2-NLS with MmImpα. These findings, together with previous results with Impα from other organisms, indicate that the differential affinity of NLSs to minor binding sites may be also responsible for the selectivity of some cargo proteins recognition and transport.

Highlights

Importin-α (Impα) is an adaptor protein that binds to cargo proteins, for their translocation to the nucleus
The crystal structures of M. musculus Impα (MmImpα) complexed to NIT-2NLS (MmImpα/NIT-2-nuclear localization sequences (NLS)) and MmImpα complexed to PAC-3-NLS (MmImpα/PAC-3-NLS) were solved at 2.15 and 1.99 Å, respectively (Table 1)
The analysis of both MmImpα/NIT-2-NLS and MmImpα/PAC-3-NLS structures showed electron densities corresponding to fragments of the peptides in two different regions of the proteins, known as major and minor binding sites (Figs. 1 and 2, respectively)

Summary

Introduction

Importin-α (Impα) is an adaptor protein that binds to cargo proteins (containing Nuclear Localization Sequences - NLSs), for their translocation to the nucleus. The NIT-2 NLS sequence binds with high affinity to the Impα major binding site from both organisms, but its binding to minor binding sites reveals interesting differences due to the presence of additional interactions of NIT-2-NLS with MmImpα. Impα is an adaptor protein that links the cargo protein to a carrier protein (importin-β; Impβ) that, through transient interactions between Impβ and NPC proteins, translocates the Impα/Impβ/cargo protein complex to the cell nucleus This process is known as the classical nuclear import pathway and is probably the most extensively used and heavily researched nuclear import mechanism[3,4,5]. A structural and calorimetric study with N. crassa Impα (NcImpα)[41] and simian virus SV40 TAg NLS resulted in higher affinity of the NLS to the minor site for N. crassa Impα than for M. musculus Impα (MmImpα)

Results

Discussion

Conclusion