Molecular Basis for the Recognition of a Nonclassical Nuclear Localization Signal by Importin β  Gino Cingolani, Janna Bednenko, Matthew T Gillespie, Larry Gerace  Molecular Cell  Volume 10, Issue 6, Pages 1345-1353 (December 2002) DOI: 10.1016/S1097-2765(02)00727-X

Figure 1 NLS Sequences and Domain Structure of Importin β (A) Sequences of cNLSs, the IBB domain of importin α, and two ncNLSs from PTHrP and ribosomal protein L5 (BIB domain). The putative phosphorylation site (Thr85) of the PTHrP-ncNLS is in red. (B) Quantitative characterization of the binding of the PTHrP-ncNLS (67–94) to wild-type importin β and to two fragments of the protein spanning HEAT repeats 1–11 and 7–19, corresponding to residues 1–485 and 304–876, respectively. The apparent dissociation constants (Kd) are shown. (C) Schematic domain organization of importin β. Each HEAT repeat is shown as two circles, corresponding to A and B helices. In green are the HEAT repeats cocrystallized with the PTHrP-ncNLS. The binding sites for the IBB domain, RanGTP, and nucleoporins are indicated by arrows. Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)

Figure 2 Overview of the Importin β (1–485):PTHrP-ncNLS (67–94) Complex (A) σA-weighted |3Fo-2Fc| electron density for the PTHrP-ncNLS (contoured at 1.15 σ) calculated using observed structure factor amplitudes, |Fo|, and calculated phases, αc, from importin β (1–485) model after rigid body refinement. The refined PTHrP-ncNLS structure, in yellow, is superimposed to the electron density. (B) Ribbon representation of the importin β (HEAT 1–11):PTHrP (67–94) complex. Importin β is ramped blue to green, and the PTHrP-NLS, in ball-and-sticks, is in yellow. Figures were generated with BOBSCRIPT (Esnouf, 1999) and Raster3D (Merritt and Bacon, 1997). Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)

Figure 3 Recognition of the PTHrP-ncNLS by Importin β (HEAT 1–11) (A) Electrostatic surface potential of importin β produced with program GRASP (Nicholls et al., 1991). The backbone of the PTHrP-ncNLS (67–94) is shown in yellow as a “worm representation,” and the three distinct moieties of the peptide are highlighted in white. (B) A summary of the interactions between importin β and the PTHrP-ncNLS. Black and purple lines indicate binding of importin β residues to side chains and main chain of the PTHrP-ncNLS, respectively. Intramolecular interactions in the PTHrP are indicated by dotted blue lines. Intra- and intermolecular contacts between atoms in distance range 2.5–4.5 Å where determined using program CNS (Brünger et al., 1998). (C) Close-up view of PTHrP-Thr85. PTHrP and importin β are in blue and yellow, respectively. Tryptophans 430 and 472 of importin β are shown in red. The dashed lines indicate the proximity from importin β Trp 430 and 472 from PTHrP-Thr85. Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)

Figure 4 Importin β Has Two Cargo Binding Sites (A) Schematic model of full-length importin β bound to both IBB domain and PTHrP-ncNLS. Only the polypeptide backbone is shown for importin β (ramped from blue to green) and PTHrP-ncNLS (in yellow). The IBB domain, in red, is shown in ribbon. The figure was generated by superimposing residues 1–485 of the structure of full-length importin β bound to the IBB domain and the complex of importin β (1–485):PTHrP (67–94). The rmsd for the 485 α carbons of the two complexes complexes is ∼1.39 Å. (B) SDS-PAGE showing the binding of PTHrP (1–108) to an importin β:IBB-β-galactosidase complex. Lane 1 shows the IBB-β-galactosidase and importin β bound to Ni-agarose beads. After addition of PTHrP (1–108) little importin β is displaced from the IBB-β-galatosidase (lane 2), while the excess of the PTHrP, shown by the asterisk, is recovered mainly in the flow-through fraction (FT, lane2) and in the subsequent wash fractions (W1 and W2, lanes 3–4). The trimeric importin β:IBB-β-galactosidase:PTHrP complex is specifically eluted by addition of 100 mM imidazole (lane 5). Lane 6 shows 10 μl of beads collected before elution, boiled for 10 min, and analyzed by SDS-PAGE. Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)

Figure 5 In Vitro Nuclear Import of BSA-PTHrP in Permeabilized HeLa Cells Nuclear accumulation is observed in the presence of full-length importin β (HEAT 1–19) (A) and a fragment comprising HEAT 1–11 (C). No significant nuclear accumulation is observed in the absence of importin β (B) or using an importin β mutant (45–485) that does not bind Ran (D). Bar, 10 μM. Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)

Figure 6 Quantification of Nuclear Import in Permeabilized Cells Nuclear import reactions were performed using three different importin β constructs (full-length [1–876], first 11 HEAT repeats [1–485], and dominant-negative mutant [Kutay et al., 1997] deficient in Ran binding [45–485]). Each import reaction was carried out at the three different concentrations of receptor. Intranuclear fluorescence intensity of approximately 100 cells was quantified using NIH Image software. Molecular Cell 2002 10, 1345-1353DOI: (10.1016/S1097-2765(02)00727-X)