The nuclear localization sequence of the epigenetic factor RYBP binds to human importin α3

Biochim Biophys Acta Proteins Proteom. 2021 Aug;1869(8):140670. doi: 10.1016/j.bbapap.2021.140670. Epub 2021 May 1.


RYBP (Ring1 and YY1 binding protein, UniProt ID: Q8N488) is an epigenetic factor with a key role during embryonic development; it does also show an apoptotic function and an ubiquitin binding activity. RYBP is an intrinsically disordered protein (IDP), with a Zn-finger domain at its N-terminal region, which folds upon binding to DNA. It is predicted that RYBP has a nuclear localization sequence (NLS), comprising residues Asn58 to Lys83, to allow for nuclear translocation. We studied in this work the ability of intact RYBP to bind Impα3 and its truncated species, ΔImpα3, without the importin binding domain (IBB), by using fluorescence and circular dichroism (CD). Furthermore, the binding of the peptide matching the isolated NLS region of RYBP (NLS-RYBP) was also studied using the same methods and isothermal titration calorimetry (ITC), and in silico molecular docking. Moreover, we carried out experiments with NLS-RYBP in the absence or in the presence of NaCl (140 mM). Our results show that RYBP interacted with Impα3 and ΔImpα3, causing protein precipitation. The NLS-RYBP also interacted with both importin species (dissociation constant in the low micromolar range), at low or high ionic strength, as shown by intrinsic fluorescence and ITC. These findings indicate that the NLS region, which was mainly unfolded in isolation in solution, was essentially responsible for the binding of RYBP to each of the importin species. Furthermore, the molecular simulations predict that the anchoring of NLS-RYBP takes place in the major binding site for the NLS of cargo proteins bound to Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in RYBP and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

Keywords: Calorimetry; Fluorescence; Nuclear location signal; Peptide; RYBP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cell Nucleus / metabolism
  • Epigenesis, Genetic / genetics
  • Epigenesis, Genetic / physiology
  • Humans
  • Intrinsically Disordered Proteins
  • Karyopherins / genetics
  • Karyopherins / metabolism
  • Magnetic Resonance Spectroscopy / methods
  • Molecular Docking Simulation
  • Nuclear Localization Signals / genetics
  • Nuclear Localization Signals / metabolism*
  • Nuclear Proteins / metabolism
  • Protein Binding / genetics
  • Protein Transport
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • alpha Karyopherins / genetics
  • alpha Karyopherins / metabolism*


  • Intrinsically Disordered Proteins
  • KPNA4 protein, human
  • Karyopherins
  • Nuclear Localization Signals
  • Nuclear Proteins
  • RYBP protein, human
  • Repressor Proteins
  • alpha Karyopherins