Nerve Growth Factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation, maintenance, growth and survival of target neurons. Expressed as a proNGF precursor, NGF is then matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct cellular partners which account for different functional roles. While the structure of mature NGF is available, little is known about the structure of the pro-domain within the

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... ext of proNGF because the dynamical and structural features of the protein have so far prevented its structure determination. We have exploited a new hybrid strategy based on nuclear magnetic resonance and modelling validated by small angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but has a clear tertiary structure propensity and forms transient tertiary intramolecular contacts. It is also able to interact, albeit weakly, with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Based on paramagnetic relaxation enhancement data and advanced molecular modelling, we have then reconstructed the overall properties of the pro-domain in the context of proNGF and showed that it has a compact structure. Our data represent an important step towards the structural and functional characterization of the properties of proNGF and its pro-domain.