1. Volume 23, Issue 2, Pages 374-384 (February 2015)
Molecular Determinants for Nuclear Import of Influenza A PB2 by Importin α Isoforms 3 and 7 
Ruth A. Pumroy, Song Ke, Darren J. Hart, Ulrich Zachariae, Gino Cingolani 
Structure 
Volume 23, Issue 2, Pages (February 2015)
DOI: /j.str
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2. Structure 2015 23, 374-384DOI: (10.1016/j.str.2014.11.015)
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3. Figure 1 Evolution and Structure of the Importin α Isoforms
(A) Phylogenetic trees of the Arm cores (left) and IBBs (right) of human importin α isoforms and the yeast importin α (Kap60).
(B) ClustalX2 alignment of the IBB region of human importin α isoforms. Basic conserved residues are in blue, with acidic residues of subfamily α3 surrounding the minor NLS in red. Highlighted in dark and light gray are identical and similar residues, respectively.
(C) Ribbon diagrams of PB2-NLD, in black, bound to the Arm core of importin α1 (right, in cyan), importin α3 (middle, in yellow), and importin α7 (left, in purple). See also Table S1.
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4. Figure 2 Structural Comparison of Human Importin α Isoforms
(A) Structural alignment of α1, α3, and α7 (shown as beads on a string) bound to PB2-NLD (in ribbons). Alignment is based on superimposition of PB2 GDs using as reference PB2-NLD solved in complex with importin α1. Beads-on-a-string models of importin α isoforms were drawn in PyMol by reducing each Arm repeat to a single point based on the α-carbon of the conserved tryptophan positioned in the middle of helix H3.
(B) Schematic diagram of the helices of an Arm repeat.
(C) Cartoon representation of the importin α isoforms with helices depicted as cylinders. Residues at the H1/H2 interface are shown as spheres, with glycines in black and polar or charged residues in green. The color coding of importin α isoforms in panels (A) and (C) is the same as in Figure 1C. See also Figure S1.
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5. Figure 3
Interactions of PB2-NLD at the Major and Minor NLS-Binding Sites of Importin α Isoforms
(A) Surface representation of importin α1 bound to PB2-NLD (shown as black ribbon); in blue are residues identical in all human isoforms; in light gray are residues that are not conserved in all human isoforms.
(B) and (C) Magnified view of the major (B) and minor (C) NLS-binding sites of importin α1 (top), importin α3 (middle), and importin α7 (bottom). See also Figure S2.
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6. Figure 4
Equilibrium Molecular Dynamics Simulations of Importin α Isoforms
End-to-end distance measurements of importin α isoforms bound to PB2-NLD (i) or free importin α1 (ii), α3 (iii), and α7 (iv). Shaded bars display equilibrium fluctuations of importin α isoforms elongations obtained from a 0.4 μs equilibrium simulation. On the y axis, P is the normalized probability distribution. See also Figure S3.
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7. Figure 5
Pulldown Analysis of the Interaction of Importin α Isoforms with NLS Cargos
GST-tagged importin α isoforms lacking the IBB (ΔIBB−) or full length (FL−) were immobilized on glutathione beads and incubated with (A) PB2-NLD, (B) NP-NLS, (C) PB2-NLD-K718A, and (D) PB2-biNLS (residues 738–759). Pulldowns are shown as mean ± SD for three experiments. Student's t test was used to determine significance, where ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, and NS is not significant. See also Figure S4.
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8. Figure 6
Pulldown Analysis of the Interaction of Importin α Isoform IBB Mutants with PB2
GST-tagged importin α1 and α3 lacking the IBB (ΔIBB−), full length (FL−), Q/R mutants, and IBB swaps were immobilized on glutathione beads and incubated with (A) PB2-NLD or (B) control NP-NLS. Pulldowns are shown as mean ± SD for three experiments. No interaction was observed between free MBP, GST-importin, and glutathione beads (Figure S5). Student's t test was used to determine significance, where ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, and NS is not significant. See also Figure S6.
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9. Figure 7 Model for PB2 Nuclear Import by Importin α Isoform 3 and 7
Schematic diagrams of (i) adaptor-independent nuclear import; (ii) adaptor and receptor-dependent nuclear import; (iii) proposed nuclear import of PB2.
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