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

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. Nucleocytoplasmic protein trafficking regulation between cell compartments is a fundamental biological process for eukaryotic organisms. The translocation of proteins across the nuclear envelope occurs through nuclear pore complexes (NPC) which, in most cases, it is an active carrier-mediated transport process 1,2. This mechanism requires additional carrier proteins or transport factors that generally belong to the β-karyopherin superfamily and specific nuclear targeting signals. The best-characterized signals are known as nuclear localization sequences (NLS), which are recognized by the importin-α protein (Impα). 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 3-5. The classical NLS (cNLS) is characterized by one or two amino acid basic clusters and is defined as monopartite or bipartite. Consensus sequences for these two cNLS were proposed and correspond to K(K/R)X(K/R) and KRX 10-12 K(K/R)X(K/R) (where X corresponds to any residue, but positively charged amino acids are preferred at this position and hydrophobic ones are also acceptable), respectively 4,6-9. These sequences bind to Impα through

Types of nuclear localization signals and mechanisms of protein import into the nucleus

“…Importins are further categorized into importin α s and importin β s , based on their structural and functional features [ 16 ]. In mammalian cells, the importin α s protein family only contains 6 members, namely, importin α1, importin α3, importin α4, importin α5, importin α6 and importin α7 [ 48 , 49 ]. The energy for nuclear transport is provided by the small Ras family GTPases.…”

Comparative study of the interaction of ivermectin with proteins of interest associated with SARS-CoV-2: A computational and biophysical approach

“…As the nuclear import for macromolecules is facilitated by importins, the structures of importin α1 subunit (PDB: 5KLR ) from Mus musculus and importin β1 subunit (PDB: 2P8Q ) from Homo sapiens were used as a model for the members of the nuclear import superfamily. The host nuclear import system can be bound and sequestered by pathogens such as SARS-CoV-2 allowing transportation of viral proteins to the host nucleus leading to increased viral replication [ [12] , [13] , [14] , [15] ]. Additionally, we also consider the multi-functional helicase (nsp13) of SARS-CoV-2 responsible for viral replication (PDB: 6ZSL ) [ [16] , [17] , [18] ], and the main protease (Mpro) of SARS-CoV-2 (PDB: 6LU7 ) as it is a key enzyme of coronaviruses and has a fundamental role in mediating viral replication and transcription, making it an attractive target for drugs [ 11 , [19] , [20] , [21] , [22] ].…”

Structural deformability induced in proteins of potential interest associated with COVID-19 by binding of homologues present in ivermectin: Comparative study based in elastic networks models

“…As the nuclear import for macromolecules is facilitated by importins, the structures of importin α1 subunit (PDB: 5KLR) from Mus musculus and importin β1 subunit (PDB: 2P8Q) from Homo sapiens were used as a model for the members of the nuclear import superfamily. The host nuclear import system can be bound and sequestered by pathogens such as SARS-CoV-2 allowing transportation of viral proteins to the host nucleus leading to increased viral replication [25] , [26] , [27] , [28] , [29] . Additionally, we also consider the multi-functional helicase (nsp13) of SARS-CoV-2 responsible for viral replication (PDB: 6ZSL) [30] , [31] , [32] , and the main protease (Mpro) of SARS-CoV-2 (PDB: 6LU7) as it is a key enzyme of coronaviruses and has a fundamental role in mediating viral replication and transcription, making it an attractive target for drugs [33] , [34] , [35] , [36] , [37] .…”