Molecular Characterisation of the Nuclear Localisation Sequence Binding Properties of Toxoplasma gondii Importin α

The classical nuclear import pathway in eukaryotic cells is mediated by the importin (IMP) α and β1 heterodimer, where IMPα acts as an adaptor protein that recognises and binds to nuclear localisation signals (NLSs) on nuclear protein cargoes. IMPα from Toxoplasma gondii (TgIMPα), the causative agent of toxoplasmosis, is an essential gene and validated druggable target, but its NLS binding properties remain largely uncharacterised. To investigate the NLS binding properties of purified TgIMPα we designed NLS peptides and compared its properties with those of mammalian (mouse) IMPα by AlphaScreen binding assays. The well-characterised viral NLS from simian virus SV40 large tumour-antigen (T-ag NLS) was recognised by TgIMPα with low-nM binding affinity comparable to Mus musculus IMPα (MmIMPα), whereas a T. gondii-specific NLS from the GCN5 gene product (TgGCN5) exhibited high affinity binding to TgIMPα but not MmIMPα. Mutational analysis of basic residues within the NLSs indicated pronounced binding selectivity. To elucidate the structural basis of these interactions, molecular dynamics (MD) simulations of an ab initio TgIMPα model bound to each NLS was performed. Key polar, salt-bridge, and cation-π interactions driving NLS recognition were identified. Critically, the lysine at position 2 (Lys128 in T-ag and Lys95 in TgGCN5) of each NLS engages conserved threonine and aspartate residues at the major binding site, playing an essential role in binding stability. Strikingly, TgIMPα displayed a more extensive and distinct cation-π interaction network than MmIMPα, revealing that, while the overall NLS binding architecture is conserved, specific pocket variations underlie differential high affinity recognition. Collectively, this study provides insights for the first time into the molecular distinctions in NLS binding between parasite and mammalian IMPα, potentially representing the foundation for future selective targeted drug design or peptidomimetic strategies against T. gondii.