Insights into the Nerve Growth Factor / endogenous ligands binding mechanism.

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types, both in the central and peripheral nervous system. Even though it was discovered almost 70 years ago, and its tertiary structure is known since 1991, many molecular and functional properties remain elusive. At the same time, its pharmaceutical interest is still high, since the protein is involved in many disease mechanisms, like chronic pain and neurodegenerative disorders. Small endogenous ligands that interact with NGF are of increasing interest, due to their likely capability of modulating its biological activity. Among these molecules, ATP was shown to mediate NGF neurotrophic activity through it receptors, TrkA and p75NTR. However, no structural information on the binding sites nor on the mechanism of the interaction is available so far. Aiming at gaining new information on this aspect, we undertook a biophysical study on NGF/ATP binding, by means of solution NMR. We have focused our studies on the recombinant human NGF (rhNGF), which is the molecule of medical interest, more that the better structural characterized mouse protein (mNGF). At first, we have obtained 15N- and 13C15N-labeled rhNGF, suitable for the NMR studies. We have optimized the protocols set up in our previous work on the mouse protein, to better adapt the expression conditions to the human protein. The 2D HSQC NMR collected spectra allowed us to identify structural features of the rhNGF in comparison to mNGF in solution. We know from our previously published data that the hNGF and mNGF do not overlap in their biochemical, biophysical and in vitro functional properties, reflected in their 3D structure in solution and highlighted by their available respective crystal structures. We therefore proceeded to the assignment of both the backbone and side chains of hNGF, to fully characterize the protein, by means of 3D NMR experiments (15N and 13C NOESYs). We investigated the binding effects of ATP and of a set of different divalent ions by means of Differential Scanning Fluorimetry and identified the suitable conditions for the NMR studies. We then moved to the investigation of the NGF/ATP binding, using a protein-based solution NMR approach. We recorded 2D HSQC spectra following a titration with increasing amounts of ATP or the more stable analogue, ATP-PCP. We could identify the binding site of ATP and the results will be described.