Structural basis of binding specificity between nuclear localization signals and importin α

Chang, Chiung-Wen (2013). Structural basis of binding specificity between nuclear localization signals and importin α PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

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Author Chang, Chiung-Wen
Thesis Title Structural basis of binding specificity between nuclear localization signals and importin α
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Bostjan Kobe
Rafael Counago
Simon Williams
Total pages 234
Total colour pages 43
Total black and white pages 191
Language eng
Subjects 0607 Plant Biology
0601 Biochemistry and Cell Biology
0699 Other Biological Sciences
Formatted abstract
In the classical nuclear import pathway, the specific recognition between the nuclear receptor (importin-α) and the nuclear localization signals (NLSs) plays an essential role for facilitating the cargo import process. Importin-α has two separate NLS-binding sites (the major and the minor sites that accommodate NLSs comprising of one (monopartite) or two clusters (bipartite) of basic residues connected by a 10 - 12 residue linker). The major NLS-binding site is the preferential binding site for most of the monopartite NLSs characterized to date, and accommodates the larger basic cluster of the bipartite NLSs. By screening random peptide libraries using importin-α variants as bait, the bound NLS sequences could be divided into six classes. The class-3 minor site-specific NLSs and class-5 plant-specific NLSs feature a shorter basic cluster. The molecular basis of the specific binding between these non-classical NLSs and importin-α was not known. In particular, there was a lack of crystal structures of plant importin-α that would explain the molecular basis of the differential binding specificity between the class-5 plant-specific NLSs and importin-α variants.

In this thesis, I investigated the binding specificity of the class-3 minor site-specific and class-5 plant-specific NLSs for importin-α variants from yeast, mammals and plants, using structural and biochemical methods. Firstly, I determined the crystal structure of rice importin-α1a (O. sativa subsp. Japonica), as the first representative structure from plants. The structure reveals a previously uncharacterized auto-inhibitory NLS-like segment in the N-terminal region of the protein, found bound at the minor NLS-binding site. Additionally, I show that the minor NLS-binding site of rice importin-α1a is a preferential binding site for the binding of both plant-specific NLSs and minor site-specific NLSs. The observation from the structures is consistent with the differential binding specificity obtained in the mutational analyses and in-vitro binding assays. Interestingly, comparative bioinformatic screens not only indicate that both plant-specific and minor site-specific NLSs are much less prevalent than the classical NLSs, but also reveal a greater prevalence of these two classes of non-classical NLSs in the rice proteome, compared to the proteomes from yeast, mammals, and even other plant species.

Furthermore, I show the distinctive binding conformation of the minor site-specific NLSs when bound to mouse importin-α. Unlike previously characterized NLSs, these NLSs form an α-helical turn, stabilized by forming internal H-bond and cation-π interactions with importin-α. This helical turn sterically hinders binding at the major NLS-binding site, explaining the minor-site preference. Together with previous studies from the literature, our data allow us to propose an optimal binding sequence ([K/R]XXKRX[F/Y/W]XXAF) for binding to the minor NLS-binding site.

Finally, I investigated the structural and biochemical basis of the specific binding between the bipartite NLSs of two virulence proteins (VirD2 and VirE2) from the plant pathogen, Agrobacterium tumefaciens, and rice importin-α1a. Unexpectedly, the VirE2 NLSs have relatively weak binding affinities for rice importin-α1a. Therefore, I only determined the crystal structures of rice importin-α1a in complex with VirD2 NLS, as well as in complex with the prototypical bipartite NLS from Xenopus nucleoplasmin. Comparison of our structures with mouse importin-α:bipartite NLS complex structures characterized previously, highlights the conservation of bipartite NLS binding modes, and distinct interactions at the linker region that differentiate the binding of bipartite NLSs to importin-α.

In summary, this thesis gives insights into a number of features of nuclear import pathway specific to plants. Combination of structural, biochemical, and bioinformatic data suggests a greater usage of the minor NLS-binding site in rice than in yeast, mammals, and even other plants species. Together with the additional autoinhibitory interactions characterized in the minor NLS-binding site, this observation may have relevance for the regulation of the import pathway in plants. Finally, the structural and biochemical studies on the binding of the VirD2, nucleoplasmin, and VirE2 bipartite NLSs to rice importin-α1a not only illustrate the conservation of the bipartite NLS binding mode on the importin-α variants, but also suggests that importin-α is unlikely to interact with the putative VirE2 NLSs. To conclude, this thesis provides advanced information on understanding the molecular basis of the specific recognition between NLSs and importin-α proteins from mammals and plants. My data can also help characterize novel cargo proteins destined for the cell nucleus by the classical nuclear import pathway.
Keyword Nuclear transport
Classical nuclear import pathway
Nuclear localization signal
Importin α
Oryza sativa (rice)
Arabidopsis thaliana

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Created: Tue, 11 Feb 2014, 12:13:25 EST by Chiung-wen Chang on behalf of Scholarly Communication and Digitisation Service