1. Volume 25, Issue 12, Pages 3371-3381.e5 (December 2018)
Structural Analyses of Toll-like Receptor 7 Reveal Detailed RNA Sequence Specificity and Recognition Mechanism of Agonistic Ligands 
Zhikuan Zhang, Umeharu Ohto, Takuma Shibata, Masato Taoka, Yoshio Yamauchi, Ryota Sato, Nikunj M. Shukla, Sunil A. David, Toshiaki Isobe, Kensuke Miyake, Toshiyuki Shimizu 
Cell Reports 
Volume 25, Issue 12, Pages e5 (December 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 3371-3381.e5DOI: (10.1016/j.celrep.2018.11.081)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Successive U-Containing ssRNAs Fully or Moderately Bound TLR7
(A) Overlay of TLR7-IMDQ-UUUUUU, TLR7-IMDQ-AAUUAA, TLR7-IMDQ-CCUUCC, TLR7-IMDQ-GGUUGG, and TLR7-IMDQ-GGUCCC complex structures. Chain A (TLR7), chain B (TLR7∗), and ssRNAs (at site 2) are indicated in pink, purple, and multiple colors, respectively, in cartoon representations. Throughout this article, asterisks are used to indicate the second TLR7 and its residues in dimeric TLR7. IMDQ (at site 1) is shown in a gray space-filling representation.
(B–F) Detailed views of ssRNAs at site 2. UUUUUU (B), AAUUAA (C), CCUUCC (D), GGUUGG (E), GGUCCC (F), and TLR7 proteins are shown in stick and surface representations, respectively. The C atoms of ssRNAs are colored differently in each figure. The O, N, and P atoms of ssRNAs are colored red, blue, and orange, respectively. Electron densities of ssRNAs contoured at the 1.0σ level (GUCCC) or 1.5σ level (except GUCCC) in the 2Fo-Fc map are displayed in gray meshes.
(G) Recognition of bases at position 1 of site 2. The water molecule is shown as a red sphere.
(H) Recognition of bases at position 3 of site 2.
(I) NFκB reporter activity for mouse or human TLR7 expressed in HEK293T cells stimulated by ssRNAs (n = 4, +SD). Two-tailed Student’s t test was used to evaluate the statistical significance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
(J) Model of the specific ssRNA sequence preference of TLR7.
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4. Figure 2 Recognition Modes of IQDs and GS9620
(A) Chemical structures of five IQDs and GS9620. R0, R1, and R3 parts of R848 and GS9620 are highlighted in green, blue, and orange, respectively.
(B) Overlay of TLR7-R848 (PDB: 5GMH), TLR7-imiquimod, TLR7-gardiquimod, TLR7-CL075, TLR7-CL097, and TLR7-GS9620 complex structures. The front view (top) and top view (bottom) are shown. TLR7 proteins are shown as a ribbon representation, and site 1 ligands are shown as a red space-filling representation. The direction of the ligand-binding pocket is indicated with a two-way arrow.
(C) Surface representations of site 1 of five TLR7-IQD complexes (left) and TLR7-GS9620 complexes (right). IQDs and GS9620 are colored blue and red, respectively. Surfaces on the left are shown for the TLR7-R848 complex (PDB: 5GMH).
(D) Detailed view of IQD recognition at site 1. Superposition of five TLR7-IQD structures is shown. The C atoms are colored sky blue (R848), green (imiquimod), yellow (Gardiquimod), pink (CL075), and slate blue (CL097). The O, N, and S atoms of IQDs are colored red, blue, and dark yellow, respectively. Hydrogen bonds are shown as dashed lines. The ligand-binding pocket formed by TLR7 and TLR7∗ is indicated schematically by thick dashed lines (right). Residues that are crucial for each key interaction are listed.
(E) Detailed views of GS9620 recognition at site 1. C atoms of GS9620 are colored gray.
(F) Superposition of TLR7-GS9620 (dark gray and magenta) and TLR7-R848 (light gray and sky blue) complexes at site 1. The proteins are omitted for clarity (right).
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5. Figure 3 Binding Analysis of Eight Site 1 Ligands
(A and B) SEC analysis of the binding between TLR7 and eight site 1 ligands in the absence (A) or presence (B) of polyU_9-mer (n = 1). The absolute elution times of TLR7, TLR7 + R848, TLR7 + polyU, and TLR7 + polyU + R848 are shown on top of each peak. Dimerization levels are indicated with a color gradation. Shifts in elution time compared with TLR7 (A) or TLR7 + polyU (B) are indicated in parentheses.
(C and D) ITC analysis of the binding between TLR7 and eight site 1 ligands in the absence (C) or presence (D) of polyU 9-mer (n = 1). KD values are shown as red characters. ND, not determined.
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6. Figure 4 Molecular Determinants of TLR7 and TLR8 Ligand Selectivity
(A) Residues involved in the recognition of site 1 ligands in TLR7 and TLR8. (Left) Sequence alignment of TLR7 (humans and monkeys) and TLR8 (humans) around site 1 (LRR8, LRR11–14, and LRR16–18), showing residues involved in the recognition of site 1 ligands highlighted in yellow (identical residues) or blue (different residues). Alignments were performed using CLUSTALW software (EMBL-European Bioinformatics Institute). (Right) Superposition of site 1 of the TLR7-G-polyU (PDB: 5GMF) and TLR8-ORN06 structures (PDB: 4R0A). TLR7, TLR8, guanosine, and uridine are colored purple, cyan, yellow, and gray, respectively. Residues selected for the generation of chimeric mutants of TLR7 and TLR8 (in parentheses) are labeled in red.
(B and C) Nuclear factor κB reporter activity for human TLR8 (B) and TLR7 (C) chimeric mutants expressed in HEK293T cells (n = 4, +SD). Two-tailed Student’s t test was used to evaluate the statistical significance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Dashed lines indicate a WT activity.
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7. Figure 5
Identification of 2′,3′-cGMP as a Possible Natural Ligand for TLR7
(A) Detailed views of 2′,3′-cGMP recognition at site 1 of the TLR7-GGUUGG complex structure. The C atoms of 2′,3′-cGMP are colored sky blue.
(B) Comparison of recognition modes of 2′,3′-cGMP and guanosine. Overlay of the structures of the TLR7-GGUUGG complex (dark gray) and the TLR7-G-polyU complex (light gray; PDB: 5GMF) at site 1 is shown. The C atoms of guanosine are colored yellow.
(C) LC-MS analysis of nucleos(t)ides in dissolved crystals. The LC elution profiles of dissolved TLR7-GGUCCC (blue line) and TLR7-GGUUGG (green line) crystals and standard injection (black line, [1] 5′-cytidine monophosphate [5′-CMP], [2] 5′-uridine monophosphate [5′-UMP], [3] cytidine, [4] 5′-GMP, [5] uridine, [6] 3′-UMP, [7] 5′-AMP, [8] 3′-GMP, [9] guanosine, [10]: 2′,3′-cGMP, [11] 2′-GMP, [12] adenosine) are shown.
(D) Nuclear factor κB reporter activity for human or mouse TLR7 expressed in HEK293T cells stimulated by various ligands (n = 6, +SD). Two-tailed Student’s t test was used to evaluate the statistical significance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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