Journal Article

Importin α3 Is Tolerant to Nuclear Localization Signal Chirality

Rizzuti, Bruno, Neira, José L., Hornos, Felipe

2025

Overview

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.

• Publisher: MDPI AG,MDPI
• Subject: alpha Karyopherins - chemistry / alpha Karyopherins - metabolism / Amino acids / Cell Nucleus - metabolism / Drugs / Enantiomers / Fluorescence / Humans / Localization / Medical research / Medicine, Experimental / Molecular Dynamics Simulation / Molecular weight / Nuclear Localization Signals - chemistry / Nuclear Localization Signals - metabolism / Polypeptides / Protein Binding / Proteins / Simulation methods / Stereoisomerism