Binding Dynamics of Isolated Nucleoporin Repeat Regions to Importin-β Timothy A. Isgro, Klaus Schulten  Structure  Volume 13, Issue 12, Pages 1869-1879 (December 2005) DOI: 10.1016/j.str.2005.09.007 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Importin-β with the Cargo SREBP-2 Fragment Bound Importin-β is shown in red, and the cargo SREBP-2 fragment is shown in blue. Importin-β HEAT repeats 10–19 are displayed in surface representation and colored differently so that they may be distinguished clearly. Note the dual α helices that compose each HEAT repeat. The A helices are typically comprised of 3–6 turns, line the outer, convex surface of importin-β, and interact with FG-Nups. The B helices are typically comprised of 3.5–8 turns and line the inner, concave surface of importin-β; the B helices interact with the signaling protein Ran and cargo, e.g., SREBP-2. Both helices are labeled in HEAT repeat 6. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Schematic View of the Binding Spots on the Surface of Importin-β All binding spots occur on hydrophobic patches of the importin-β surface. The N and C termini are indicated along with HEAT repeats 1–19. Binding spots identified by the current molecular dynamics simulations are labeled in gray, while those spots that were known experimentally before the study are labeled with a black slash. Binding spots that were identified with the conservation criterion described are labeled with a black dot. Each binding spot is labeled with a number. The molecular dynamics simulations reproduced the experimental binding spots (with the exception of the experimentally weak spot between HEAT repeats 15/16) and predicted several new binding spots. Binding spot #4 was identified experimentally after the completion of this study (Liu and Stewart, 2005). Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Views of Three Experimental Binding Spots that Have Been Verified in the Current Study (A and B) (A) Binding spot #2 (HEAT 5/6) at the completion of molecular dynamics simulation 4, compared with the (B) experimental structure. The simulation reveals residue aF1 binding for 26.3 ns to a conserved pocket formed by Val221, Gln220, Phe217, Ile178, and Arg182. The experimental structure shows the binding of only core FSFG residues of a chain of sequence DDSKPAFSFGAAAA for comparison with the simulated adduct. Note that the experimental structure shows the peptide residue F9 binding to the “left” side of Phe217. (C and D) (C) Binding spot #3 (HEAT 6/7) at the completion of molecular dynamics simulation 1, compared with the (D) experimental structure. The simulation reveals residues qF7 and qL6 binding to a conserved pocket formed by importin-β residues Ala259, Cys223, Met219, and Tyr255 for 46.6 ns. The experimental structure shows the binding of residue F6 from the peptide AAAAAFSF to the same pocket. (E) Binding spot #7 (HEAT 14/15) at the completion of simulation 3. Importin-β residues Leu612, Tyr646, Met608, Ala609, and Ala649 form the hydrophobic and conserved pocket that binds residue jF5 for 26.1 ns. The simulation provides a structure for this adduct. The importin-β surface is colored by residue conservation and hydrophobicity; red indicates completely conserved and hydrophobic residues, and blue indicates nonconserved residues. See “Sequence Conservation” for details. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Nonbonded Binding Energies over the Course of Simulation for Three Representative Binding Spots: #1, #2, and #4 Binding energies were calculated between the binding FG-Nup peptides and any residues on the surface of importin-β within 5 Å of any atom in the peptide. (A) Van der Waals (vdW) energy for the binding spots plotted as a function of simulation time. Binding spot #1 is shown in blue, #2 is shown in red, and #4 is shown in yellow. Note the drop in vdW energy upon initiation of each binding spot at ∼18, 10, and 15 ns, respectively. (B) Electrostatic energy for the binding spots plotted as a function of simulation time. Data are shown in gray and black. Running averages over 2 ns shown in blue, red, and yellow (color coding as in [A]) are used to show the trend in electrostatic energy during binding, which closely mimics that of the vdW energies. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 Alignment of HEAT Repeats 14/15 with 16/17 (A) The structure of both HEAT repeats at the end of simulation 4. HEAT repeats 14/15 are shown in green, while 16/17 are shown in blue, with their appropriate residues shown in atom type coloring (carbon, light blue; oxygen, red; nitrogen, dark blue; sulfur, yellow). Residues Tyr646, Leu612, Phe643, Lys645, Met608, Asp605, Leu601, and Glu642 in HEAT repeats 14/15 correspond well structurally to residues Tyr731, Leu697, Phe728, Lys730, Met693, Asp690, Leu686, and Glu727 in HEAT repeats 16/17. (B) Both HEAT repeats after structural alignment by using STRIDE via the VMD Multiple Alignment plugin. After alignment, the backbone rmsd of both pairs of HEAT repeats is 2.21 Å, and the rmsd of the eight residues shown, excluding hydrogen atoms, is 2.16 Å with respect to one another. Hydrogens have been removed for clarity. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 6 Views of Four Conserved Binding Spots Revealed by the Simulations (A) Binding spot #1 (HEAT 3/4) at the completion of simulation 4. Residue aF3 binds to a conserved pocket formed by Leu96 for 10.4 ns. (B) Binding spot #5 (HEAT 8/9) at the completion of simulation 1. The simulation reveals residue hF5 binding to a hydrophobic channel formed on one side by importin-β residues Phe370 and Tyr382 for 34.2 ns. (C) Binding spot #9 (HEAT 16/17) at the completion of simulation 3. Residues bbF7 and bbL6 bind to a conserved pocket formed by importin-β residues Tyr731, Leu697, Phe728, Lys730, Met693, and Asp690. The pocket is very similar to binding spot #7 between HEAT repeats 14/15. (D) Binding spot #10 (HEAT 16/17) at the completion of simulation 3. Residue dL11 binds into a deep “side” pocket (between the convex and concave faces of importin-β) for 12.3 ns. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 7 Sequence Conservation of Importin-β (A) Alignment of importin-β sequences from eight species. A subset of the complete sequence (residues 292–438) is displayed for clarity. Each residue is given a score from 0 to 100 based on similarity and conservation; data are displayed in a bar graph. A series of Gonnet matrices was used to score the alignment. Hydrophobic residues are colored light blue, while Phe and Tyr are colored yellow. (B) Sequence conservation of residues 292–438 mapped onto the surface of importin-β. Red indicates residues that are completely conserved across all eight species and are also hydrophobic, while blue indicates residues that are not conserved and are dissimilar across species. FG-Nup binding spots are hypothesized to occur at regions of high conservation and hydrophobicity on the importin-β surface. Binding spot #5 is indicated. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 8 Views of Two Hydrophobic Binding Spots, Not Rigorously Conserved, Revealed by the Current Simulations Hydrophobic residues are shown in white, while all other importin-β residues are shown in orange. (A) Binding spot #4 (HEAT 7/8) at the completion of simulation 1. The simulation reveals residue lF5 binding to a pocket formed by Tyr321, Ile265, and Phe261 for 31.3 ns. (B) Binding spot #6 (HEAT 11/12) at the completion of simulation 3. The simulation reveals residue iF5 binding to a pocket formed by Ala450, Leu453, Leu505, Ile457, Ser502, and the side chain of Gln454 for 14.9 ns. Structure 2005 13, 1869-1879DOI: (10.1016/j.str.2005.09.007) Copyright © 2005 Elsevier Ltd Terms and Conditions