title: Recognition of human tumor necrosis factor α (TNF-α) by therapeutic antibody fragment: energetics and structural features.
abstract: Human tumor necrosis factor α (TNF-α) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-α results in the development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-α recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied the energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-α by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-α is an enthalpy-driven process due mainly to specific noncovalent interactions taking place at the TNF-α-Fab binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by entropically favorable solvation contributions. Moreover, the three-state model analysis of TNF-α unfolding shows that at physiological concentrations, TNF-α may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-α concentration could have a considerable effect on the TNF-α activity. We believe that this study sets the energetic basis for understanding of TNF-α inhibition by antibodies and its unfolding linked with the concentration-dependent activity regulation.
authors.affiliation: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia.
authors.forename: Jaka
authors.initials: J
authors.lastname: Marušič
authors.forename: Črtomir
authors.initials: Č
authors.lastname: Podlipnik
authors.forename: Simona
authors.initials: S
authors.lastname: Jevševar
authors.forename: Drago
authors.initials: D
authors.lastname: Kuzman
authors.forename: Gorazd
authors.initials: G
authors.lastname: Vesnaver
authors.forename: Jurij
authors.initials: J
authors.lastname: Lah
mesh_terms: D000915:Antibody Affinity / Q000502:physiology*; D001666:Binding Sites, Antibody; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology / Q000627:therapeutic use; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000276:immunology
chemical_list: D007140:Immunoglobulin Fab Fragments; D014409:Tumor Necrosis Factor-alpha