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. 2020 Jun 4;15(6):e0231542.
doi: 10.1371/journal.pone.0231542. eCollection 2020.

Unveiling functional motions based on point mutations in biased signaling systems: A normal mode study on nerve growth factor bound to TrkA

Pedro Túlio Resende-Lara  1   2 , David Perahia  2 , Ana Lígia Scott  1 , Antônio Sérgio Kimus Braz  1
Affiliations

Unveiling functional motions based on point mutations in biased signaling systems: A normal mode study on nerve growth factor bound to TrkA

Pedro Túlio Resende-Lara et al. PLoS One. .

Abstract

Many receptors elicit signal transduction by activating multiple intracellular pathways. This transduction can be triggered by a non-specific ligand, which simultaneously activates all the signaling pathways of the receptors. However, the binding of one biased ligand preferentially trigger one pathway over another, in a process called biased signaling. The identification the functional motions related to each of these distinct pathways has a direct impact on the development of new effective and specific drugs. We show here how to detect specific functional motions by considering the case of the NGF/TrkA-Ig2 complex. NGF-mediated TrkA receptor activation is dependent on specific structural motions that trigger the neuronal growth, development, and survival of neurons in nervous system. The R221W mutation in the ngf gene impairs nociceptive signaling. We discuss how the large-scale structural effects of this mutation lead to the suppression of collective motions necessary to induce TrkA activation of nociceptive signaling. Our results suggest that subtle changes in the NGF interaction network due to the point mutation are sufficient to inhibit the motions of TrkA receptors putatively linked to nociception. The methodological approach presented in this article, based jointly on the normal mode analysis and the experimentally observed functional alterations due to point mutations provides an essential tool to reveal the structural changes and motions linked to the disease, which in turn could be necessary for a drug design study.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Multisignaling receptors and biased signaling.
The binding of a nonspecific ligand (blue sphere) to a multisignaling receptor promotes a change in the receptor’s dynamics. This change results in motions that are linked to α and β signaling pathways (brown and purple shapes, respectively). On the other hand, a biased ligand (purple hexagon) is able to activate a singular signaling path (β, orange shape). A mutation in the receptor can also result in biased signaling. In this figure, the interaction of a nonspecific ligand with the mutant receptor results in the activation of β signaling path (blue shapes), but not the α one. In addition, the presence of nonfunctional motions are observed in all interactions.
Fig 2
Fig 2. NGF/TrkA-Ig2 complex and binding sites.
(A) Domain organization of TrkA. Domains are colored in: orange, cysteine-rich clusters (C1-2); in magenta, leucine-rich repetitions (LRR1-3); in green, immunoglobulin-like domains (Ig1-2); in red, transmembrane portion (TM); and in blue, tyrosine kinase domain. (B) NGF/TrkA-Ig2 complex motif description. (C) Specific and (D) conserved NGF/TrkA epitopes. Chains are colored as: NGFA, green; NGFB: cyan; TrkAA: magenta; TrkAB: yellow.
Fig 3
Fig 3. TrkA intracellular signaling activated by wild type and mutant NGF.
(Left panel) Wild type signaling. NGF (green cartoon) promotes TrkA (blue cartoon) signaling mediated by phosphorylation of intracellular tyrosine residues (dark blue boxes). Phosphorylated Y496 forms an adaptor binding site that couple mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K) signaling pathways, activating transcription of genes related to neuronal differentiation and survival, respectively. Phosphorylation of Y791 activates phospholipase Cγ1 (PLCγ1) pathway, that mediates neuronal differentiation and synaptic plasticity via PKC and Ca+2 cytosolic release, respectively. Autophosphorylation of residues Y676, Y680 and Y681 mediates TrKA kinase activity and modulates Y496 and Y791 signaling. (Right panel) R221W mutant signaling. R221W mutation (orange spheres in NGF cartoon) incapacitates the induction of Y791 signaling pathway, impairing neuronal differentiation and synaptic plasticity, that results in the insensitivity to pain, heat and cold in HSAN5.
Fig 4
Fig 4. Representation of neurotrophic and nociceptive related motions.
(A) Frontal and (B) bottom view of WT mode 11, that show NGF torsional motions approaching TrkA-Ig2 subunits. NGF dimer is represented in transparent cartoon, and TrkA is represented by secondary structure colored cartoon. Black arrows are normal mode eigenvectors placed at Cα atoms.
Fig 5
Fig 5. Motion redundancy among the 20 lowest frequency normal modes computed for TrkA complexed with wild type and mutant NGF.
The mantel similarity scores revealed that a number of WT and R221W normal modes (red and green nodes, respectively) preserve a given motion. Thus, since both WT and mutant NGF are capable to induce neurotrophic response, these similar modes are putatively assigned to activate the neurotrophic bias of TrkA. On the other hand, independent WT motions (blue nodes), that have no similarity either with all other WT motions or R221W ones, are hypothesized as responsible to nociceptive bias of TrkA, since they are present only in WT structures. Also, independent R221W motions (magenta nodes) may introduce an impairment in communication pathways. Numbers in circles are non-trivial NM numbers of respective systems. Values on edges correspond to the Mantel similarity scores between the connected modes; only pairs of modes with similarity score greater than |0.6| were connected in these graphs. Note that the modes considered start with the number 7, since the modes from 1 to 6 correspond to trivial overall translation/rotation of the system.
Fig 6
Fig 6. Dynamic Cross-Correlation Map (DCCM) of atomic displacements for all residue pairs in different types of motions.
(A) DCCM of WT QNTR motions. TrkA chains show moderate anti-correlation between them (green square). The NGFB and TrkAB chains have strongly anti-correlated residues (black rectangle). (B) DCCM of QNCP motions. TrkA chains show a more consistent moderate correlation with each other (green square), indicating that they move in a coordinate fashion in the same direction. The same was observed for NGF. Also, more strongly anti-correlated residues were observed involving all chains (black rectangle). (C) The DCCM of the R221W QNTR motions have a very similar pattern to that of the WTNTR. (D) The DCCM of the QMUT motions. Residue pair correlations are weaker throughout the structure in these motions. TrkA chains are uncoordinated (green square).
Fig 7
Fig 7. Community analysis reveals differences in the dynamic coupling of the complex.
Mapping of the molecular structure of network communities (colored regions) and residue couplings (lines) of (A) WT QNTR, (B) R221W QNTR, (C) QNCP, and (D) QMUT. QNTR motions shows the coupling of communities in specific and conserved binding sites. In particular, in the two WT networks, the NGF main community (blue) links TrkA subunits with a similar strength while those in the mutants exhibit asymmetric behavior, coupling TrkAB with much more strength than TrkAA. Left panel: the relative radius of circles indicates the number of residues in a particular community. The values on the edges correspond to the relative strength of inter-community coupling. Right panel: the magenta circles highlight the dynamic coupling of residues H205 (NGFB) and Q350 (TrkAB), and the green circles indicate the coupling of F133 (NGF) and H297 (TrkA) in the two chains. Inter-community couplings are indicated with black lines.
Fig 8
Fig 8. Differences in residue-wise centralities in dynamical networks.
(A) Comparison between WT QNTR and R221W QNTR residue centralities indicates great similarity all along the complex. (B) Despite the similar centrality values for both TrkA chains, QNCP and QMUT present remarkable differences at NGF interface regions. Purple and green stripes indicate specificity and conserved patch residues, respectively. Arrows indicates residues with high degeneracy in interface regions. Black connectors represent disulfide bonds.
Fig 9
Fig 9. Optimal and suboptimal paths of interface residues.
Optimal and suboptimal paths coupling the TrkA residues (A) E339A–E339B, (B) H297A–H297B, (C) Q350A–Q350B and (D) H297A–Q350B. QNTR motions can couple the two binding sites, while QNCP motions efficiently connect TrkA chains only via specific patch. The QMUT suboptimal paths are longer in almost all cases, which indicates its contribution to communication impairment. The shortest path is shown in the representation of the structure. Source and sink residues are presented as licorice. The histograms show the length distribution of the 500 paths calculated for each pair of residues.
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This work was performed using HPC resources from the “Mésocentre” computing center of Centrale Supélec and École Normale Supérieure Paris-Saclay supported by Centre Nationale de Recherche Scientifique (CNRS, France) and Région Île-de-France (http://mesocentre.centralesupelec.fr/). We gratefully acknowledge financial support from Universidade Federal do ABC (UFABC) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), process number: 88881.133141/2016-01 to PTRL.