Molecular Characterisation of the Nuclear Localisation Sequence Binding Properties of Toxoplasma gondii Importin α
- 1Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
- 2Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Monash, Clayton, VIC 3800, Australia
Abstract
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.
Related
- Molecular and Structural Characterisation of the MERS-Coronavirus Polymerase
- Molecular epidemiology of Toxoplasma gondii in impala (Aepyceros melampus) from the Greater Kruger in South Africa: Detection of the Africa 4 lineage
- Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis
- Spectral characterisation of short-wave infrared (SWIR) tissue chromophores and tissue-mimicking phantom optical properties
- Molecular Characterisation of the Romanian African Swine Fever Virus p72 and B602L Genetic Markers.
BibTeX
@article{mehta2026molecular,
title = {{Molecular Characterisation of the Nuclear Localisation Sequence Binding Properties of Toxoplasma gondii Importin α}},
author = {Diksha Mehta and Barnava Banerjee and Fasséli Coulibaly and Kylie M. Wagstaff and Swati Patankar and Prasenjit Bhaumik and David A. Jans},
journal = {Cells},
year = {2026},
month = oct,
doi = {10.3390/cells15201861},
url = {https://www.mdpi.com/2073-4409/15/20/1861},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Molecular Characterisation of the Nuclear Localisation Sequence Binding Properties of Toxoplasma gondii Importin α
AU - Diksha Mehta
AU - Barnava Banerjee
AU - Fasséli Coulibaly
AU - Kylie M. Wagstaff
AU - Swati Patankar
AU - Prasenjit Bhaumik
AU - David A. Jans
PY - 2026
DA - 2026/10/13
JO - Cells
DO - 10.3390/cells15201861
UR - https://www.mdpi.com/2073-4409/15/20/1861
AB - 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.
SN - 2073-4409
ER -