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Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1

Ho Yee Joyce Fung1, Yuh Min Chook2

  • 1Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 12, 2022
PubMed

Insights

This study details a protocol for purifying proteins and forming a ternary complex for X-ray crystallography. This method aids in understanding the CRM1 nuclear export receptor and its interactions.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • The Karyopherin protein CRM1 (also known as XPO1) is a key regulator of nuclear export for cellular macromolecules.
  • CRM1 functions by recognizing nuclear export signals (NESs) on cargo proteins.
  • Previous studies utilized engineered ternary CRM1-Ran-RanBP1 complexes for structural determination via X-ray crystallography.

Purpose of the Study:

  • To present a detailed protocol for the purification of individual proteins involved in CRM1-mediated nuclear export.
  • To describe the formation of a ternary CRM1-Ran-RanBP1 complex suitable for structural studies.
  • To outline the crystallization process for this complex to enable X-ray crystallography.

Main Methods:

  • Protein purification techniques for individual components.
  • In vitro assembly of the ternary CRM1-Ran-RanBP1 complex.
  • Crystallization screening and optimization for X-ray diffraction.

Main Results:

  • Successful purification of individual CRM1, Ran, and RanBP1 proteins.
  • Formation of a stable ternary CRM1-Ran-RanBP1 complex.
  • Development of a reproducible crystallization protocol for the ternary complex.

Conclusions:

  • The presented protocol facilitates the structural investigation of CRM1-mediated nuclear transport.
  • This method provides a foundation for studying CRM1 interactions with NES-containing cargoes and inhibitors.
  • The optimized crystallization approach aids in determining high-resolution structures of CRM1 complexes.
Keywords:
CRM1KPTLMBLeptomycin BNESNuclear export signalsSINEX-ray crystallographyXPO1

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