1. Volume 28, Issue 1, Pages 57-67 (October 2007)
Structural Basis for Substrate Recognition and Dissociation by Human Transportin 1 
Tsuyoshi Imasaki, Toshiyuki Shimizu, Hiroshi Hashimoto, Yuji Hidaka, Shingo Kose, Naoko Imamoto, Michiyuki Yamada, Mamoru Sato 
Molecular Cell 
Volume 28, Issue 1, Pages (October 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Sequence Alignment of Five Known NLSs with PY Motif
For each NLS, three consensus residues (H/R, P, and Y) recognized in site A and one hydrophobic or basic residue (V/A/P/K) in site B are colored red. hnRNP A1 NLS is also called M9 NLS.
Molecular Cell  , 57-67DOI: ( /j.molcel )
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3. Figure 2 Structures of Trn1
(A) Overall structures of NLS (substrate)-free Trn1 (red) and Trn1 bound to hnRNP D NLS (green), TAP NLS (blue), and JKTBP NLS (yellow), where α helices are represented as cylinders. Twenty consecutive HEAT repeats (H1–H20), each of which is composed of two antiparallel helices, are labeled on the NLS-free Trn1 structure.
Each H8 loop is shown as a black cylinder (α helix) and a black dotted line (disordered region). Fo − Fc map contoured at 2.5 σ for each NLS is also shown.
(B) The CNS composite simulated annealing omit map of the NLS region bound to Trn1. The map was calculated with coefficient 2Fo− Fc and contoured at 1.0 σ. NLSs of hnRNP D, TAP, and JKTBP are shown as stick models colored green, blue, and yellow, respectively.
(C) Overall structure of Trn1-Ran complex, where α helices are represented as cylinders and the H8 loops are colored black. Structure was drawn with the refined coordinates deposited in the Protein Data Bank (accession code 1QBK).
(D) Trn1 structure showing the conformational changes upon either RanGTP or NLS binding. The structures of NLS-free Trn1 (red), Trn1 bound to hnRNP D NLS (green), Trn1 bound to TAP NLS (blue), Trn1 bound to JKTBP NLS (yellow), and Trn1-RanGTP complex (brown) were superimposed in the overlapping region of the N- and C-terminal arches (HEAT repeats 8–13). The 20 consecutive HEAT repeats in each Trn1 molecule are represented by straight lines. Structure of Trn1-RanGTP complex was drawn with the refined coordinates deposited in the Protein Data Bank (accession code 1QBK).
(E) Schematic illustration of Trn1 structure and domain organization. Two antiparallel helices (A and B helices) in each HEAT repeat are colored orange and yellow, respectively. The H8 loop (residues 312–374) defined as the long loop connecting the H8A and H8B helices is colored black.
Molecular Cell  , 57-67DOI: ( /j.molcel )
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4. Figure 3 NLS Recognition by Trn1
(A) Schematic illustrations of Trn1 interactions with hnRNP D NLS (left) and TAP NLS (right). Trn1-NLS contacts less than 3.8 Å are shown. HEAT repeats 8–13 correspond to site A, and HEAT repeats 14–18 to site B.
(B) Structures of hnRNP D NLS (green), TAP NLS (blue), hnRNP A1 NLS (orange), and hnRNP M NLS (purple) bound to Trn1. Two close-up views of the structures at the right side and one close-up view at the left side show the interactions with Trn1 at sites A and B, respectively. Structures of hnRNP A1 NLS and hnRNP M NLS bound to an H8 loop-truncated Trn1 mutant were drawn with the refined coordinates deposited in the Protein Data Bank (accession codes 2H4M and 2OT8).
Molecular Cell  , 57-67DOI: ( /j.molcel )
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5. Figure 4
Effect of Site A and Site B Mutation on In Vitro Nuclear Transport
Digitonin-permeabilized cells were incubated with 1 μM GST-GFP-hnRNP D NLS for 20 min at 30°C in the presence of (A) 0.5 μM Trn1 wild-type, (B) 0.5 μM Trn1 W373A (NLS-binding site unrelated mutant), (C) 0.5 μM Trn1 W460A, (D) 0.5 μM Trn1 E509A, and (E) 0.5 μM Trn1 W730A.
Molecular Cell  , 57-67DOI: ( /j.molcel )
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6. Figure 5 Effect of H8 Loop Mutation on NLS Dissociation
Interaction of Trn1 or H8 loop mutant R343A/V345G with various GST-tagged NLSs shown on the left lower corners of the gels in the presence and absence of RanGppNHp and RanGDP was studied by GST pull-down assay. Signs + and + + for RanGppNHp indicate, respectively, the first and second treatments of a bead-bound form of GST-NLS-Trn1 complex with RanGppNHp.
Molecular Cell  , 57-67DOI: ( /j.molcel )
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7. Figure 6
Schematic Illustration of a Proposed Mechanism for the Nuclear Import Pathway Mediated by Trn1
The Trn1 molecule is represented by the S-like cyan ribbon labeled with N and C termini. NPC is the nuclear pore complex. Red and blue ellipsoids on the NLS in transport substrate show the three consensus residues (red ellipsoid) and one hydrophobic residue (blue ellipsoid).
Molecular Cell  , 57-67DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions