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. 2017 Oct 9;8(1):810.
doi: 10.1038/s41467-017-00864-2.

Structural Basis for IL-1α Recognition by a Modified DNA Aptamer That Specifically Inhibits IL-1α Signaling

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Free PMC article

Structural Basis for IL-1α Recognition by a Modified DNA Aptamer That Specifically Inhibits IL-1α Signaling

Xiaoming Ren et al. Nat Commun. .
Free PMC article

Abstract

IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.The cytokine interleukin 1α (IL-1α) plays an important role in inflammatory processes. Here the authors use SELEX to generate a modified DNA aptamer which specifically binds IL-1α, present the structure of the IL-1α/aptamer complex and show that this aptamer inhibits the IL-1α signaling pathway.

Conflict of interest statement

A.D.G., I.v.C. and N.J. are employees and shareholders of SomaLogic, Inc. The remaining authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Structural and schematic representations of the IL-1α/SL1067 complex and SL1067. a Overall structure of the IL-1α/SL1067 complex. IL-1α is colored green and SL1067 is colored cyan with the 2Nap modified residues colored orange. b Sequence alignment of IL-1α and IL-1β. IL-1α residues that are involved in the non-polar and polar interactions with SL1067 are denoted with black stars and triangles separately. c Side view of the SL1067 structure. The Nap modified nucleotides are colored orange. d Schematic representation of SL1067. The three parts of SL1067: stem, turn, and loop are marked. All the base pairs are labeled according to the nomenclature of Leontis et al.
Fig. 2
Fig. 2
Important structural motifs in SL1067. a Overall structure of SL1067 with the nucleotides that participate in the formation of three important motifs colored as mentioned below. The zipper motif is indicated by a green oval. b The dU3-dA16=dU8 triple is colored pink. c The G-quadruplex is colored magenta. d The bottom dA5–dA18 pair is colored blue
Fig. 3
Fig. 3
Key elements of the SL1067 structure. a The snap formed by 2Nap15 and the sugar ring of dG2 combines the stem and loop regions of SL1067. b Multilayer stacking of 2Nap7, the base of dU10 and the base of dA9 is capped with O4ʹ of dU8. c O4ʹ(dG13) caps the Nap-base stack between dU10 and dU14. d The hydrophobic cluster on SL1067. The dU7 and 2Nap8 residues form a classical edge-to-face π interaction. Two other pairs of edge-to-face π interactions are formed between 2Nap10 and 2Nap15, and 2Nap14 and dG11. 2Nap8 makes face-to-face stacking with 2Nap10. e Four-layer base stacking is capped with the O4ʹ of the dU15 sugar ring on the top and the O4ʹ of dA5 sugar ring on the bottom. The stacking is further strengthened by the base pairings of dA9-dU15, dA16-dU8 and dG17-dG6. f In the loop region, the dG12 and dG13 base rings form face-to-face stacking and dG11 protrudes toward a hydrophobic pocket on the IL-1α surface
Fig. 4
Fig. 4
The hydrophobic interactions along the IL-1α/SL1067 interface. a The IL-1α surface is transparent. The SL1067 residues that are evolved in the hydrophobic contacts with IL-1α are rendered as sticks. b The Ile68 side chain and 2Nap7 form a stacking interaction similar to Ile18 and 2Nap14. c The Met15 side chain forms edge-to-face π interactions with 2Nap10 and 2Nap15, separately. The Arg16 side chain forms edge-to-edge contacts with 2Nap10 and 2Nap15, respectively. Modified nucleotides are labeled in black and amino acid residues forming the binding interface are labeled in gray throughout. The two gray arrows indicate the direction and degree of rotation for each inset view
Fig. 5
Fig. 5
The H-bond network along the IL-1α/SL1067 binding interface is mediated by a Na+ ion and three water molecules. a W1, W2, W3, OD1(Asp64), O(Asp65), and O21(2Nap-dU7) are coordinated by a sodium ion. Additional H-bonds are formed with the surrounding residues from either IL-1α or SL1067: W1-O (Asp64), W2-O2 (2Nap-dU10), O(Ala66)-W3-O2(dU10). The lengths of these H-bonds are listed in Supplementary Tables 4 and 5. b The same view of the H-bond network as shown in a. 2Fo–Fc electron density for the Na+ ion and surrounding water molecules is shown in gray mesh and contoured at 1σ. c Wall-eyed stereo image of the IL-1α/SL1067 crystal structure. Electron density from the final 2Fo–Fc map contoured at 1.0σ. SL1067 is colored orange and the protein is colored in green. Red spheres depict associated water molecules
Fig. 6
Fig. 6
Polar interactions at the binding interface. a The Watson–Crick face of dU7 participates in two H-bonds with Ser61. An additional H-bond with Lys60 is mediated by W4. b The Watson–Crick face of dG11 forms H-bonds with Ile68 and Trp113. c W5 mediates hydrogen bonding between Arg16 and 2Nap-dU14
Fig. 7
Fig. 7
Determination of the binding affinity of SL1067 to IL-1α and the competition of IL-1α receptors with SL1067. a Shown is a four-parameter global fit (mean ± S.D., n = 4) of the relative fraction bound vs. IL-1α concentration. The mean (±S.D.) binding dissociation constant (K d) determined from the four independent experiments is 7.3 ± 1.5 nM. b Displacement of radiolabeled SL1067 by unlabeled competitors SL1067 (open circles), IL-1 RI (open squares) or IL-1 RII (open triangles). Shown is a global fit (mean ± S.D., n = 3) to a one-site competition model of competitor concentration vs. fraction no competitor. The mean (±S.D.) inhibitory constants (K i) from three determinations are: SL1067, K i = 4.3 ± 1.5 nM; IL-1 RI K i = 21 ± 9.8 nM and IL-1 RII K i = 290 ± 130 nM
Fig. 8
Fig. 8
A cell-based assay shows the inhibition of IL-1α downstream signaling by SL1067 in a dose-dependent manner. Shown are the global fits (mean ± S.D., n = 3) to a four-parameter sigmoidal dose response model of the modified aptamer concentration vs. the cytokine secretion normalized to the percent of control (no modified aptamer condition). a, b SL1067 inhibits the secretion of IL-6, a downstream effector of IL-1 receptor signaling in HS27 (open circles) and HuVEC (open diamonds). The mean (±S.D.) half minimal inhibitory concentration (IC50) from three replicates are 22 ± 1.8 nM and 2.7 ± 0.73 nM in HS27 and HuVEC cells, respectively. c, d SL1067 inhibits the secretion of IL-8, another downstream effector of IL-1 receptor pathway in HS27 (open circles) and HuVEC (open diamonds). The IC50 (±S.D.) from three replicates are 23 ± 3.6 nM and 3.5 ± 0.70 nM in HS27 and HuVEC cells, respectively

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