Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

Abstract

Nerve growth factor-β (NGF) is essential for the correct development of the nervous system. NGF exists in both a mature form and a pro-form (proNGF). The two forms have opposing effects on neurons: NGF induces proliferation, whereas proNGF induces apoptosis via binding to a receptor complex of the common neurotrophin receptor (p75NTR) and sortilin. The overexpression of both proNGF and sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, whereas the structure of NGF has been determined by X-ray crystallography, the structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure motif in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two -linked and two-linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.

The structure of the Pro-domain of mouse proNGF in contact with the NGF domain

“…Our data are consistent with our previous Surface Plasmon Resonance (SPR) binding studies of proNGF towards the NGF antibodies alphaD11 and 4C8, which show a reduced affinity with proNGF as compared to NGF (Paoletti et al, 2009). They also justify a reduced affinity of proNGF to both TrkA and p75NTR receptors (Paoletti et al, 2009) and indicate a conformational heterogeneity of the pro-domains in each proNGF in contrast to the previously reported models based on HDX-MS data (Trabjerg et al, 2017).…”

“…This region is the same that was demonstrated in the classic paper by Suter et al (1991) to be necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF. Our data also agree with HDX-MS experiments (Trabjerg et al, 2017) which have suggested that residues R81-F89 change the structural dynamics of loops I (G144-E156), II (A161-F170) and V (D214-A219). However, in contrast with the HDX-MS experiments, our models propose the occurrence of an interaction between the exposed W142 of NGF with the pro-domain residues Y21-E29, as previously reported (Paoletti et al 2011;Paoletti et al 2009;Kliemannel et al 2007).…”

“…Of the two trajectories, trajectory A visits more helical conformers (average helicity 31% with a standard deviation of 28%). Interestingly, in both trajectories we frequently observe close contacts between residues R81-F89 of the proNGF pro-domain and loops I, II and IV in the NGF part (Figure S5F), in agreement with recent H/D exchange experiments (Trabjerg et al, 2017). A full dimer structure extracted from the simulations in which one pro domain satisfies >80% of the NGFpdS24C-MTSL contacts and the other >80% of the NGFpdS90C-MTSL contacts is shown in Figure 7C, while the E34-R49 region is in a helical conformation (its position is marked by a red cross in Figure 7B).…”

“…Finally, we noticed the absence in the proNGF spectrum of a set of resonances belonging to the central stem of the NGF domain (F174, F175, T177, V208, K209 and A210). This suggests a substantial change of chemical environment of the NGF domain within proNGF induced by the pro-domain, with residues R81-F89 causing a structural stabilization of loops I, II and V of NGF in agreement with recent HDX-MS findings (Trabjerg et al, 2017). Altogether the data are fully consistent with the experimentally based models.…”

“…Fluorescence and H/D exchange measurements indicated contacts between W142 of the NGF domain with residues W37-A57 of the proNGF pro-domain (Kliemannel et al, 2007). It was also recently reported that the pro-domain in proNGF induces a structural stabilization of loops I, II and IV in the NGF part, suggesting a direct interaction between residues R81-F89 of the proNGF pro-domain and loops I, II and IV in the NGF part (Trabjerg et al, 2017).…”