title: Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal.
abstract: 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.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Carolin
authors.initials: C
authors.lastname: Koerner
authors.forename: Tinglu
authors.initials: T
authors.lastname: Guan
authors.forename: Larry
authors.initials: L
authors.lastname: Gerace
authors.forename: Gino
authors.initials: G
authors.lastname: Cingolani
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D017433:Protein Structure, Secondary; D051381:Rats; D014364:Tryptophan; D028961:beta Karyopherins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019913:Nuclear Localization Signals; D028961:beta Karyopherins; D014364:Tryptophan