Conformational Rigidity within Plasticity Promotes Differential Target Recognition of Nerve Growth Factor

Abstract

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early '90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody αD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

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

“…The resonances of residues belonging to loop III (A181-V189) were instead negligibly affected but had different NOE contacts. These findings confirmed the plasticity of the NGF loops (Paoletti et al, 2016) also in the context of proNGF. 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).…”

“…EOM analysis of proNGF in buffer yielded high quality fit with χ 2 value of 1.48 (Figure 8A, curve 2). Minor deviations at higher angles (s > 0.20 Å -1 ) can be explained by the presence of different conformations of NGFpd (Paoletti et al, 2016). The preponderant fraction of models in the optimized ensemble have Rg between 2.8-3.2 nm (Figure 8A, insert).…”

“…NGFpd mutants were expressed and purified as for the wild-type. 15 N and 2 H doubly labelled proNGF was expressed, refolded and purified according to a previous protocol (Paoletti et al, 2016), but growing the cells in perdeuterated water.…”

Biomimetic surface delivery of NGF and BDNF to enhance neurite outgrowth

“…(k) BDNF release profile from loaded surface within 168 hr. Adaptation from Paoletti et al (2016) and Robinson et al (1999) [Color figure can be viewed at wileyonlinelibrary.com]…”

Generation of rationally-designed nerve growth factor (NGF) variants with receptor specificity

“…In support of this, FoldX predicted that substitution of H4 residue of NGF with D or E amino acid will alter TrkA affinity. The N-terminal domain of NGF is not observed in the co-crystal structure with p75 NTR due to conformational flexibility and thus difficult to resolve by x-ray crystallography, and as such is not thought to contribute to binding of p75 NTR [19]. The predicted changes in free energy of binding of the NGF variant-receptor complex (ΔΔG) and on protein stability are shown in Fig.…”