期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 23, 页码 -出版社
MDPI
DOI: 10.3390/ijms232315333
关键词
nuclear localization signal; binding; biolayer interferometry; calorimetry; fluorescence; molecular docking
资金
- La Ligue Contre le Cancer
- INCa
- Canceropole PACA
- INSERM
- Comunidad Valenciana
- [CAICO 2021/0135]
This study investigated the binding ability of NLS-Myc peptide to Imp alpha 3 and Delta Imp alpha 3 through experiments and simulations. The results showed that NLS-Myc can interact with both importin species, and there are some unique properties in the binding process, such as the difference in the release mechanism depending on the presence or absence of the IBB domain.
The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin alpha 3 (Imp alpha 3) and its truncated species (Delta Imp alpha 3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of similar to 0.5 mu M (for Imp alpha 3) and similar to 60 nM (for Delta Imp alpha 3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Imp alpha 3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Imp alpha 3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain.
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