title: New binding mode to TNF-alpha revealed by ubiquitin-based artificial binding protein.
abstract: A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins--designed ankyrin repeat proteins--without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hoffmann
authors.forename: Michael
authors.initials: M
authors.lastname: Kovermann
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Markus
authors.initials: M
authors.lastname: Fiedler
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Sven
authors.initials: S
authors.lastname: Pfeifer
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002151:Calorimetry; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D009682:Magnetic Resonance Spectroscopy; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin