Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal

J Biol Chem. 2003 May 2;278(18):16216-21. doi: 10.1074/jbc.M301137200. Epub 2003 Feb 19.


Molecular recognition of the importin beta-binding (IBB) domain of importin alpha by importin beta is critical for the nuclear import of protein cargoes containing a classical nuclear localization signal. We have studied the function of four conserved tryptophans of importin beta (Trp-342, Trp-430, Trp-472, and Trp-864) located at the binding interface with the IBB domain by systematic alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent when analyzed individually. Interestingly, the combination of the hot spot at residue Trp-864 with mutations in the other three tryptophans gives rise to a striking synergy that diminishes IBB domain binding by up to approximately 1000-fold and, in turn, abolishes import activity. We propose that importin beta uses the tryptophans to select and stabilize a helical conformation of the IBB domain, which, in turn, conveys specific, high affinity binding.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Active Transport, Cell Nucleus
  • Animals
  • Binding Sites
  • Cell Line
  • Mutagenesis, Site-Directed
  • Nuclear Localization Signals*
  • Protein Structure, Secondary
  • Rats
  • Tryptophan
  • beta Karyopherins / chemistry*
  • beta Karyopherins / metabolism


  • Nuclear Localization Signals
  • beta Karyopherins
  • Tryptophan