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.
Highlights
Lized cells by importin  [6, 7]
We have studied the function of four conserved tryptophans of importin  (Trp342, Trp-430, Trp-472, and Trp-864) located at the binding interface with the IBB domain by systematic alanine substitution mutagenesis
Conserved Tryptophans of Importin  at the Binding Interface with the IBB Domain—One of the intriguing features of the importin 1⁄7I〉〉-binding interface is the presence of four conserved tryptophans in importin , Trp-342, Trp-430, Trp472, and Trp-864 (Fig. 1a), which are located in close proximity (3.8 – 4.2 Å) to four highly conserved IBB residues [9], namely Lys-18/22 (Fig. 1, b and c) and Arg-13/51 (Fig. 1, d and e)
Summary
Lized cells by importin  [6, 7]. Translocation of the import complex through the nuclear pore complex (NPC) involves multiple rounds of interaction of importin  with nucleoporins [8]. The binding cavity of importin  is highly enriched in acidic residues and affords an ideal environment for the folding of a basic peptide This has led to the idea that importin  may serve both as an import receptor and a chaperone to keep small basic proteins from aggregating [12]. We have demonstrated that this hot spot can synergize with mutations in other tryptophans at the binding interface, which only marginally affect the interaction when analyzed alone. Based on these data, we propose a model for recognition of the IBB domain by importin  that involves the selection of a population of distinct helical IBB conformers by the receptor
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