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

The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its Nuclear Localization Sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3 the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.