ATP binding to the Nerve Growth Factor: a molecular switch for neurotrophins signaling?

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types. Despite its discovery in the '50s, many molecular and functional properties remain elusive. Small endogenous NGF ligands with biological modulating effect are of increasing interest. Among these, ATP showed to mediate NGF neurotrophic activity through its receptors, although the determinants of the binding mechanism are yet to be unraveled. We thus undertook a biophysical study on NGF/ATP binding. We obtained 15N- and 13C15N-labeled recombinant human NGF (rhNGF), through the optimization of protocols previously established for mouse NGF (rmNGF). Interestingly, rhNGF and rmNGF differ in both their biochemical/biophysical and functional properties, despite their highly similar 3D structures. We performed 2D and 3D NMR experiments in order to complete the backbone and side chains resonance assignment of rhNGF. Differential Scanning Fluorimetry was used to investigate the binding effects of ATP and of a set of divalent ions on rhNGF. We verified by FT-IR that NGF secondary structure is retained in presence of ATP. We then moved to the investigation of the rhNGF/ATP binding mode, using protein-based solution NMR. We recorded 2D HSQC spectra following a titration with increasing amounts of ATP. We identified the likely binding site of ATP on rhNGF dimer and NMR guided molecular docking unveiled a possible interaction mechanism. Finally, we investigated the ATP binding mode of ATP on rhNGF by means of Isothermal Titration Calorimetry. In order to suggest an effect of ATP binding on the NGF/receptors signaling pathways, we undertook a study on the receptor binding by means of SPR. The effect of different divalent cations was also taken into consideration, and the obtained results are interestingly related to the role of NGF and proNGF in health and disease.