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. 2021 May 7:19:2938-2949.
doi: 10.1016/j.csbj.2021.05.009. eCollection 2021.

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions

Francesca Paoletti  1 , Franci Merzel  1 , Alberto Cassetta  2 , Iza Ogris  1 , Sonia Covaceuszach  2 , Jože Grdadolnik  1 , Doriano Lamba  2   3 , Simona Golič Grdadolnik  1
Affiliations

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions

Francesca Paoletti et al. Comput Struct Biotechnol J. .

Abstract

The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF's role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

Keywords: ARIA, Ambiguous Restraints for Iterative Assignment; ATP modulation; BDNF, Brain Derived Neurotrophic Factor; CARA, Computer Aided Resonance Assignment; CS-E, Chrondroitin Sulfate E; CSP, Chemical Shift Perturbation; DSF, Differential Scanning Fluorimetry; EI-MS, Electron Ionization Mass Spectrometry; Endogenous ligands; FGF2, Fibroblast Growth Factor 2; FT-IR, Fourier Transform Infrared Spectroscopy; HBD, Heparin Binding Domain; HSQC, Heteronuclear Single Quantum Coherence; ITC, Isothermal Titration Calorimetry; MALDI-TOF MS, Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry; MD, Molecular Dynamics; MS, Mass Spectrometry; NGF interactions; NGF, Nerve Growth Factor; NMR, Nuclear Magnetic Resonance; NOE, Nuclear Overhouser Effect; NOESY, Nuclear Overhauser Effect Spectroscopy; NT, NeuroTrophin; Neurotrophins; P20, Polysorbate 20; PME, Particle Mesh Ewald; RMSD, Root Mean Square Deviation; SAR, Structure-Activity Relationship; SPR, Surface Plasmon Resonance; STD, Saturation-Transfer Difference; TrkA, Tyrosine Kinase Receptor A; TrkA, p75NTR receptors; p75NTR, p75 NeuroTrophin Receptor; proNGF, proNGF – NGF precursor; rh-proNGF, recombinant human proNGF – NGF precursor; rhNGF, recombinant human NGF; rmNGF, recombinant mouse NGF.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
3D structure of rhNGF in solution. a) Overlay of the 20 lowest global energy models after ARIA refinement in explicit water. The protein backbone is shown as ribbon. The two protomers are colored in green and magenta, respectively. b) Cartoon model of the medoid solution NMR structure of rhNGF (PDB: 6YW8). c) Cartoon model of the medoid solution NMR structure of rhNGF (blue; PDB: 6YW8) superimposed with the X-ray crystal structure of rhNGF (red; PDB ID: 1WWW). Loops have been labelled according to MacDonald et al. . Figure produced using PyMOL . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Comparison of the 3D NMR solution structures of rhNGF and rmNGF: MD results. (a, b) RMSD per residue averaged over the 300 ns MD simulation of rhNGF (PDB: 6YW8) and rmNGF (PDB: 5LSD) versus residue position for the two protomers (purple—protomer 1; green—protomer 2). (c, d, e, f, g, h) MD Evolution of the distances (Å), along the 300 ns MD simulation, between atoms engaged in the hydrogen bonds that stabilize the helical structure of the N-terminus of rhNGF (c, e, g) or rmNGF (d, f, h) (blue—protomer 1; red—protomer 2). Atoms pairs analyzed are indicated on the panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
ATP-rhNGF interactions by NMR titration. (a) – Map of combined 1H/15N CSP (Δδ) of the HN groups of rhNGF upon binding of ATP. Only values above the threshold of 0.02 ppm are shown, based on the overall shift variation due ligand’s addition. Residues with largest CSP (Δδ ≥ 0.04 ppm) are labelled. The combined 1H/15N CSP (Δδ) were calculated from 1H and 15N CSP using the equation: Δδ = ((Δδ 1H)2 + (0.04 × Δδ 15N)2)1/2. (b, c) – Expanded regions of 2D 1H-15N HSQC spectra showing a larger shift of A97 (b) and I104 (c) during titration (Colors for different ATP:rhNGF ratios: Red: 0 eq; Orange: 5 eq; Green: 10 eq; Cyan: 15 eq; Violet: 20 eq). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Binding orientation of ATP on rhNGF. (a, b) – Representation of the most frequently occupied regions for ATP onto rhNGF, according to the MD simulations (yellow spheres clusters). Sites 1 and 2 are indicated by red and black circles, respectively. Superposition of the clusters on rhNGF (blue) and the TrkA-d5 domain (green, PDB: 1WWW) (a) and p75NTR extracellular domain (magenta, PDB: 1SG1) (b). (c, d) – Representative poses from MD analysis for Site 1 (c) and Site 2 (d). Cartoon transparent blue: rhNGF; cartoon transparent purple: residues with larger CSP; ATP is represented as colored by element (C – green; N – blue; O – red; H – white; P – orange). Residues V20, E55 and F49 showing new NOEs upon ATP binding are labelled and colored by element (C – magenta; N – blue; O – red; H – white). The distances between HN protons of rhNGF and ATP protons (indicated by a blue arrow) are represented by black broken lines. Figures produced using PyMOL . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
rhNGF ligands binding sites. rhNGF residues in contacts with ATP during MD simulations (green filled diamonds). rhNGF residues affected in NMR CSP upon ATP titration (red filled circle). rhNGF residues predicted by computational docking to be involved (within 5 Å of the oligosaccharide) in CS-E binding (light blue filled “stars”); rhNGF residues within 5 Å from ATP binding Site 1 or Site 2 according to the best MD poses (see Fig. 4) are highlighted by magenta or yellow upper lines, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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