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.
Highlights
Human TNF-␣ is a cytokine involved in many disease-related cellular processes
The calorimetric biding isotherms accompanying association of fragment of adalimumab (Fab) to TNF-␣ exhibit the characteristics of a 3:1 association process (Fig. 2). This observation is confirmed by a very good fitting of the family of the isothermal titration calorimetry (ITC) curves measured at different T with the corresponding model function based on the binding model that assumes the existence of three equivalent independent Fab binding sites on the TNF-␣ molecule (Fig. 2; see the supplemental material for details)
In line with the ITC data are the results of the circular dichroism (CD) spectroscopic measurements, which clearly indicate that the association of TNF-␣ with Fab is accompanied by significant rearrangements of Fab and/or TNF-␣ structure (Fig. 2)
Summary
Human TNF-␣ is a cytokine involved in many disease-related cellular processes. Results: High affinity binding of therapeutic antibody (inhibitor) to native and molten globule-like TNF-␣ conformation is driven by specific noncovalent interactions. The availability of certain biopharmaceuticals to bind TNF-␣ and block its binding to receptors enables successful treatment of these pathologies Such TNF-␣ inhibitors are in most cases monoclonal antibodies, e.g. infliximab, adalimumab, golimumab; some other approaches to TNF-␣ inhibition are effective as well [14]. Because quantitative thermodynamic analysis and structural modeling of binding events in such a complex interacting system are not possible, we simplified the process by using a fragment of adalimumab (Fab) instead of the full-length antibody. Thermodynamic parameters obtained from global analysis of experimental data measured at various conditions (temperature, protein concentration, and urea concentration) are discussed in terms of structural alterations that accompany the observed binding and unfolding processes. We designed a structural model of the TNF-␣-Fab complex that was, in the absence of its three-dimensional structure, used in molecular interpretation of the obtained thermodynamic parameters of binding (Fig. 1)
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