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

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

Bending-Twisting Motions and Main Interactions in Nucleoplasmin Nuclear Import.

Alpha solenoid proteins play a key role in regulating the classical nuclear import pathway, recognizing a target protein and transporting it into the nucleus. Importin-α (Impα) is the solenoid responsible for cargo protein recognition, and it has been extensively studied by X-ray crystallography to understand the binding specificity. To comprehend the main motions of Impα and to extend the information about the critical interactions during carrier-cargo recognition, we surveyed different conformational states based on molecular dynamics (MD) and normal mode (NM) analyses. Our model of study was a crystallographic structure of Impα complexed with the classical nuclear localization sequence (cNLS) from nucleoplasmin (Npl), which was submitted to multiple 100 ns of MD simulations. Representative conformations were selected for calculating the 87 lowest frequencies NMs of vibration, and a displacement approach was applied along each NM. Based on geometric criteria, using the radius of curvature and inter-repeat angles as the reference metrics, the main motions of Impα were described. Moreover, we determined the salt bridges, hydrogen bonds and hydrophobic interactions in the Impα-NplNLS interface. Our results show the bending and twisting motions participating in the recognition of nuclear proteins, allowing the accommodation and adjustment of a classical bipartite NLS sequence. The essential contacts for the nuclear import were also described and were mostly in agreement with previous studies, suggesting that the residues in the cNLS linker region establish important contacts with Impα adjusting the cNLS backbone. The MD simulations combined with NM analysis can be applied to the Impα-NLS system to help understand interactions between Impα and cNLSs and the analysis of non-classic NLSs

Geometric analysis of the bending and twisting motions of Imp<i>α</i>.

<p>(A) The bending motion was quantitatively characterized by the radius of curvature along NM7 (solid line) and PC1 (dashed line) for Imp<i>α</i> -NplNLS and along NM7 for Apo Imp<i>α</i> (dotted line), whereas (B) the twisting motion was quantitatively characterized by the average values for the angles between helices along NM9 (solid line) and PC3 (dashed line) for Imp<i>α</i> -NplNLS and along NM9 for Apo Imp<i>α</i> (dotted line).</p

The starting structure of Imp<i>α</i>-NplNLS for MD simulations.

<p>(A) The Imp<i>α</i> as a cartoon diagram colored based on each ARM repeat as a rainbow spectrum from N-terminal (blue) to C-terminal (red) and the NplNLS as a cyan cartoon diagram positioned in an antiparallel configuration compared to Imp<i>α</i>. (B) The surface representation of Imp<i>α</i> with the NplNLS as a cyan stick diagram, indicating both major (blue) and minor (orange) binding sites. (C) The major site zoom indicating positions P2–P5 and (D) the minor site zoom in P1’ and P2’. In both sites, the positively charged side chains are positioned in the main pockets of the Imp<i>α</i> binding core.</p

Schematic representation of the interactions observed in the Imp<i>α</i>-NplNLS interface.

<p>(A) The major (P2–P5) and minor (P1’-P2’) sites and the linker region are indicated in the sequence of the NplNLS. (B) The standard MD (300 ns ensemble) and (C) NM-displacement (ensemble from reference structures 67,730 ps, 207,080 ps and 274,970 ps) interaction scheme is shown with salt bridges (red) and hydrogen bonds (green) as dashed-lines, and hydrophobic contacts are shown as arcs with radiating spokes. The important tryptophan residues that mediate the hydrophobic contacts are depicted in the scheme as black sticks. The main chain of the NplNLS is represented as a gray horizontal line with its respective amino acid sequences, together with side chains shown as perpendicular lines. Only interactions that had an occupancy rate ≥50% of the analyzed trajectories are indicated in the scheme.</p

Schematic representation of the interactions observed in the Imp<i>α</i>-NplNLS interface.

<p>(A) The major (P2–P5) and minor (P1’-P2’) sites and the linker region are indicated in the sequence of the NplNLS. (B) The standard MD (300 ns ensemble) and (C) NM-displacement (ensemble from reference structures 67,730 ps, 207,080 ps and 274,970 ps) interaction scheme is shown with salt bridges (red) and hydrogen bonds (green) as dashed-lines, and hydrophobic contacts are shown as arcs with radiating spokes. The important tryptophan residues that mediate the hydrophobic contacts are depicted in the scheme as black sticks. The main chain of the NplNLS is represented as a gray horizontal line with its respective amino acid sequences, together with side chains shown as perpendicular lines. Only interactions that had an occupancy rate ≥50% of the analyzed trajectories are indicated in the scheme.</p

Main motions observed from NM analysis of Imp<i>α</i> -NplNLS.

<p>Imp<i>α</i> (cartoon model) is shown as a rainbow spectrum from N-terminal (blue) to C-terminal (red), and NplNLS (cyan cartoon model) is positioned in an antiparallel configuration compared to Imp<i>α</i>. The vector arrows for NM7–9 are shown with the correspondent description of the motion that they described. NM7 is shown in a front view, whereas NM8 and NM9 are shown in an upper view (90° rotation in the X-axis).</p