Hornos Felipe, Rizzuti Bruno, Neira José L
IDIBE, Instituto de investigación, Desarrollo e Innovación en Biotecnologia Sanitaria de Elche, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain.
CNR-NANOTEC, Institute of Nanotechnology of Consiglio Nazionale delle Ricerche, SS Rende (CS), Department of Physics, University of Calabria, 87036 Rende, Italy.
Int J Mol Sci. 2025 Aug 13;26(16):7818. doi: 10.3390/ijms26167818.
Several carrier proteins are involved in nuclear translocation from the cytoplasm to the nucleus in eukaryotic cells. We have previously demonstrated the binding of several intact folded and disordered proteins to the human isoform importin α3 (Impα3); furthermore, disordered peptides, corresponding to their nuclear localization signals (NLSs), also interact with Impα3. These proteins and their isolated NLSs also bind to the truncated importin species ∆Impα3, which does not contain the N-terminal disordered importin binding domain (IBB). In this work, we added a further 'layer' of conformational disorder to our studies, testing whether the isolated D-enantiomers of NLSs of selected proteins, either folded or unfolded, were capable of binding to both Impα3 and ∆Impα3. The D-enantiomers, like their L-form counterparts, were monomeric and disordered in isolation, as shown by nuclear magnetic resonance (NMR). We measured the ability of such D-enantiomeric NLSs to interact with both importin species by using fluorescence, biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and molecular simulations. In all cases, the binding affinities were within the same range as those measured for their L-isomer counterparts for either Impα3 or ∆Impα3, and the binding locations corresponded to the major NLS binding site of the protein. Thus, the stereoisomeric nature is not important in defining the binding of proteins to the main component of classical cellular translocation machinery, although the primary structure of the hot-spot site for NLS binding of importin is well defined.
几种载体蛋白参与真核细胞中从细胞质到细胞核的核转运过程。我们之前已证明几种完整的折叠蛋白和无序蛋白与人源亚型输入蛋白α3（Impα3）结合；此外，与其核定位信号（NLS）相对应的无序肽也与Impα3相互作用。这些蛋白及其分离出的NLS也与截短的输入蛋白物种∆Impα3结合，∆Impα3不包含N端无序的输入蛋白结合结构域（IBB）。在这项工作中，我们在研究中增加了另一层构象无序，测试所选蛋白的NLS的分离D型对映体，无论其折叠与否，是否能够与Impα3和∆Impα3结合。如核磁共振（NMR）所示，D型对映体与其L型对应物一样，单独存在时是单体且无序的。我们通过荧光、生物层干涉术（BLI）、等温滴定量热法（ITC）和分子模拟来测量此类D型对映体NLS与两种输入蛋白物种相互作用的能力。在所有情况下，对于Impα3或∆Impα3，其结合亲和力与针对其L型异构体对应物测得的亲和力处于相同范围内，且结合位置对应于该蛋白的主要NLS结合位点。因此，立体异构性质在定义蛋白质与经典细胞转运机制主要成分的结合方面并不重要，尽管输入蛋白NLS结合热点位点的一级结构已明确界定。
