9clg
Importin alpha isoform 2 with synthetic zero net-charge nuclear localization signal
Structural highlights
FunctionIMA1_MOUSE Functions in nuclear protein import as an adapter protein for nuclear receptor KPNB1. Binds specifically and directly to substrates containing either a simple or bipartite NLS motif. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Publication Abstract from PubMedThe nucleus, as the control center of the eukaryotic cell, is a prime target for therapeutic interventions due to its role in regulating genetic material. Importin-alpha is critical for successful nuclear import as it recognizes and binds to cargo proteins bearing a classical nuclear localization signal (NLS), which facilitates their transport from the cytoplasm into the nucleus. NLS tagging to 'actively' import therapeutics provides the most effective means of maximizing nuclear localization and therapeutic efficacy. However, traditional NLSs are highly cationic due to the recognition and binding requirements with importin-alpha. Because of their highly 'super-charged' nature, NLS-tagged therapeutics face significant challenges, including poor pharmacokinetics due to non-specific interactions. In this study, we engineered novel NLS tags with zero net charge to potentially overcome this limitation. Computational modeling and experimental validation revealed that these net-neutral NLSs bind to importin-alpha with similar modes and energies as their cationic counterpart. High-resolution structural determination and analysis by X-ray crystallography then confirmed their binding modes. Biophysical methods using circular dichroism, microscale thermophoresis, and cellular localization studies demonstrated that these NLSs maintain sufficiently stable complexes and acceptable binding to importin-alpha and are functional. Additionally, this study revealed that the minor NLS-binding site of importin-alpha, with its extensive cationic surface area, was particularly suited for interactions with the acidic residues of the net-neutral NLSs. This study provides a foundational understanding of NLS-importin interactions and presents net-neutral NLSs as viable candidates for next-generation NLS-therapeutic development and expands the scope of nuclear-targeting therapies. Probing the extent of importin-alpha targeting of the TAF8 NLS by eliminating its cationic net-charge.,Abdoli A, Yang Z, Odeh-Ahmed A, Bednova O, Lemieux B, Dawe L, Ravel-Chapuis A, Lavigne P, Zeytuni N, Leyton JV Protein Sci. 2025 Sep;34(9):e70272. doi: 10.1002/pro.70272. PMID:40852899[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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