Crystal Structure of Interleukin-6 in Complex with a Modified Nucleic Acid Ligand

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (K_d = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (K_d = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (K_d = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.     

Leucine-rich Repeat 11 of Toll-like Receptor 9 Can Tightly Bind to CpG-containing Oligodeoxynucleotides, and the Positively Charged Residues Are Critical for the High Affinity

TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.