The binding of ATP to the Nerve Growth Factor: a new mechanism for neurotrophins signaling.

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types. It was discovered in the '50s [1], and its tertiary structure is known, but many molecular and functional properties remain elusive. Small endogenous NGF ligands are of increasing interest, due to their likely capability of modulating its biological activity. Among these, ATP was shown to mediate NGF neurotrophic activity through its receptors, TrkA and p75NTR [2]. However, no molecular information on the binding mechanism is available. We undertook a biophysical study on NGF/ATP binding, by means of solution NMR. We obtained 15N- and 13C15N-labeled rhNGF, through the optimization of the protocols set up for the mouse protein [3]. We know from our previous publications that hNGF and mNGF do not overlap in their biochemical/biophysical and functional properties, reflected in their 3D structure in solution and in crystal. We collected 2D HSQC NMR spectra and then proceeded to the assignment of backbone and side chains of hNGF, by means of 3D NMR experiments (15N- and 13C-NOESYs). We investigated the binding effects of ATP and of a set of divalent ions by means of Differential Scanning Fluorimetry. We then moved to the investigation of the NGF/ATP binding, 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 NGF dimer and performed molecular docking to propose an interaction mechanism. Based on this we suggest an effect of ATP binding on the NGF/receptors interaction.