Abstract
The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Å2 of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters 17RAKKMSK23 and 3KR4, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala18 between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.
Highlights
A distinguishing feature of all eukaryotic cells is the containment of their genetic material within a stable and segregated nuclear organelle
Interaction with the nuclear localisation signals (NLSs) generally occurs at the concave face of the ARM domains, at locations that are typically driven by the type of NLS; monopartite NLSs bind at the major NLS-binding site, and bipartitite NLSs, comprised of two positively charged separated by a 10–12 residue linker, bind by spanning both the major and minor sites of IMPa [7,8,9]
The usual nomenclature for describing the interactions between IMPa and NLSs [10,11] designates residues binding in the minor site as P19, P29 etc., and residues binding the major site as P1, P2 etc
Summary
A distinguishing feature of all eukaryotic cells is the containment of their genetic material within a stable and segregated nuclear organelle. Following rigid body refinement and simulated annealing, analysis of both 2Fo–Fc and simulated annealed omit maps revealed clear density in the major and minor NLS-binding sites of IMPa, enabling residues of the Prp20 NLS to be built (Fig. 2).
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