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

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          Abstract

          Background: Traditional aptamers favor polar interactions with protein binding partners.

          Results: The IL-6·SOMAmer structure reveals an interface rich in hydrophobic interactions that overlap the binding sites of IL-6 receptors.

          Conclusion: Hydrophobic modifications on DNA scaffolds generate diverse and novel structural motifs.

          Significance: Synthetic SOMAmers are potent, specific, and chemically versatile ligands with distinct binding properties compared with conventional aptamers.

          Abstract

          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 n m) 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 n m). 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 n m). 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.

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          Most cited references27

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          Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6.

          The STAT family of proteins carries out a dual function: signal transduction and activation of transcription. A new family member, Stat3, becomes activated through phosphorylation on tyrosine as a DNA binding protein in response to epidermal growth factor (EGF) and interleukin-6 (IL-6) but not interferon gamma (IFN-gamma). It is likely that this phosphoprotein forms homodimers as well as heterodimers with the first described member of the STAT family, Stat91 (renamed Stat1 alpha), which is activated by the IFNs and EGF. Differential activation of different STAT proteins in response to different ligands should help to explain specificity in nuclear signaling from the cell surface.
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            Stability and kinetics of G-quadruplex structures

            In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.
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              Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130.

              Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R), the cDNA of which has already been cloned. We report here that an 80 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37 degrees C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0 degree C. Human IL-6-R can associate with a murine gp130 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                21 March 2014
                12 January 2014
                12 January 2014
                : 289
                : 12
                : 8720-8734
                Affiliations
                From the []SomaLogic, Inc., Boulder, Colorado 80301,
                [§ ]Emerald Bio, Bainbridge Island, Washington 98110, and
                the []Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan
                Author notes
                [2 ] To whom correspondence should be addressed: SomaLogic, Inc., 2945 Wilderness Place, Boulder, CO 80301. Tel.: 303-625-9020; Fax: 303-449-1057; E-mail: njanjic@ 123456somalogic.com .
                [1]

                These authors contributed equally to this work.

                Article
                M113.532697
                10.1074/jbc.M113.532697
                3961693
                24415767
                aab85663-f1ed-400e-b516-2ec32417cb6b
                © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Unported License applies to Author Choice Articles

                History
                : 12 November 2013
                : 9 January 2014
                Categories
                Protein Structure and Folding

                Biochemistry
                aptamers,dna structure,drug discovery,interleukin,molecular evolution,nucleic acid chemistry,protein-nucleic acid interaction,g-quartet,selex

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