Intrinsic structural disorder of mouse proNGF

Abstract

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X-ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75(NTR) receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular "crab-like" and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the "crab-like" to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75(NTR) receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

Nerve growth factor regulates axial rotation during early stages of chick embryo development

“…The early expression of NGF and its receptors suggested a potential developmental function in the chicken embryo. To address this question, HH 11-12 chicken embryos were injected in ovo with the anti-NGF monoclonal antibody (mAb) αD11 (15)(16)(17). This antibody has a much higher affinity for mature NGF than for unprocessed proNGF and specifically neutralizes NGF activity in vitro and in vivo by preventing binding to both TrkA and p75 NTR receptors.…”

“…This dose dependence might be explained by the differential binding properties of the anti-NGF mAb αD11, characterized by a 2,000-fold higher binding affinity for mature NGF than for proNGF (17). We can assume that the same binding selectivity would be observed for chicken NGF, because the amino acid NGF sequences of the mAb αD11 epitope are identical in chicken, mouse, and human NGF (Fig.…”

Ligand signature in the membrane dynamics of single TrkA receptor molecules

“…In vitro proNGF shows a lower binding affinity to TrkA than NGF (supplementary material Fig. S2) (Paoletti et al, 2009;Covaceuszach et al, 2010); consistently, in vivo proNGF induces lower receptor phosphorylation levels than NGF (Fahnestock et al, 2004). We find that proNGF induces a lower number of TrkA receptor trajectories populating regions 5 and 2 (Fig.…”

The role of nerve growth factor and its receptors in tumorigenesis and cancer pain

“…However, recent studies showed that proNGF, the precursor form of NGF, is really largely exist in central nervous system tissues, and has biological functions exceeding its role as a precursor (17,28,29). ProNGF could induce cell death by binding to p75NTR with high affinity (30)(31)(32). But it shows low affinity to TrkA, another receptor of NGF (33).…”