Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

<div><p>Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of <i>αβ</i>-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.</p></div

Energy profiles at longitudinal inter-dimer interface.

<p>The figures show the sum of energetic contributions of residues located at distance intervals of 3 Å apart, plotted against the radial distance between these residues and the MT lumen (A, B, C) or the tangential distance between the residues and laterally adjacent dimer (D, E, F) in GTP- and GDP-Model. Dotted lines represent the center of mass of tubulin. (A) Radial distribution of total energy in both models. Blue dashed arrow shows how destabilizing the effect of Mg²⁺ is on the GDP-Model if it remains after GTP hydrolysis. (B) Radial distribution of the energy components <i>E</i>(vdW), <i>E</i>(ele+GB) and <i>E</i>(SA) in the GDP-Model and in (C) the GTP-Model, (D) Tangential distribution of total energy in both models. On the <i>x</i>-axis, <i>x</i> < 30 is the intermediate domain and <i>x</i> > 30 is the nucleotide binding domain. (E) Tangential distribution of the energy components <i>E</i>(vdW), <i>E</i>(ele+GB) and <i>E</i>(SA) in the GDP-Model and in (F) the GTP-Model.</p

Mechanism of MT disassembly.

<p>(A) Radial energy distribution of the GDP-Model at the longitudinal inter-dimer interface is superposed on a protofilament to show how uneven the energy distribution is. This produces a torque that leads to outward curling of the protofilament. (B) Tangential energy distribution of the GDP-Model showing slight sideway tilting due to the slightly uneven distribution of energy. <i>α</i>-subunits are colored blue while <i>β</i>-subunits are red.</p

A matrix showing individual contributions of each subunit to longitudinal stability in the two simulated systems, in kcal/mol.

<p>A matrix showing individual contributions of each subunit to longitudinal stability in the two simulated systems, in kcal/mol.</p

Domain contributions to overall energy.

<p>Energetic contributions of important domains across lateral interface in (A) <i>α</i> and (B) <i>β</i> subunits and across longitudinal inter-dimer interface in (C) <i>α</i> and (D) <i>β</i> subunits. Data are shown for GDP- and GTP-Model as well as the difference between them (GTP-GDP). On the <i>x</i>-axis of (A) and (B), domains H4 helix and before occur at lateral interface of the ligand while domains after that occur at receptor lateral interface. In (C), all domains belong to receptor while all the domains in (D) belong to ligand. See ligand and receptor definitions in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004313#pcbi.1004313.g003" target="_blank">Fig 3</a>.</p

A matrix showing individual contributions of each subunit to lateral stability in the two simulated systems, in kcal/mol.

<p>Cell “L<i>β</i>”, for example, refers to the contribution of the <i>β</i> subunit of the dimer acting as ligand.</p><p>A matrix showing individual contributions of each subunit to lateral stability in the two simulated systems, in kcal/mol.</p

Energy diagrams of the complete MT ring.

<p>The diagram shows the magnitude of favorable interaction energies at each interface between two tubulin dimers, whether at (A) the lateral interface, or at (B) the longitudinal inter-dimer interface. The magnitude of the interactions is proportional to the swelling at each interface with swellings in (A) being exaggerated to aid viewing. Green lines represent GTP-Model while red lines represent GDP-Model.</p

Model of MT structure.

<p>(A) A model of an MT lattice showing <i>α</i> (blue) and <i>β</i> (red) tubulin subunits. It shows the plus and minus end as well as the seam. (B) A model of the system used in the molecular dynamics simulations. Tubulin dimers are numbered from 1 to 13, GDP (or GTP) cofactor is shown in green within <i>β</i>-tubulin, the second GTP cofactor is buried between <i>α</i> and <i>β</i> subunits, water is represented by the white box, within which purple spheres represent Cl⁻ and brown spheres represent Na⁺. Periodic box dimensions in units of Å are also shown.</p

Energetic contributions of residues.

<p>Difference between overall residual contributions per MT ring in GTP- and GDP-Model; (</p><p></p><p></p><p></p><p><mi>E</mi><mi>i</mi>GTP</p><mo>−</mo><p><mi>E</mi><mi>i</mi>GDP</p><p></p><p></p><p></p>), where <i>i</i> is the residue number running from 1 to 1742. Different energy axes are used due to differences in magnitude of interactions at both interfaces. Important residues are labeled together with their domains.<p></p

Tubulin subsystems used for MM/GBSA calculations.

<p>(A) A subsystem of two lateral tubulin dimers extracted from MT simulations where the receptor (<i>R</i>) is tubulin <i>k</i> and the ligand (<i>L</i>) is tubulin <i>k</i>+1, where <i>k</i> runs from 1 to 12, (B) Same as (A) but at the seam. I.e. the receptor is tubulin 13 and the ligand is the periodic image of tubulin 1. Residue (Res.) numbering in (A) and (B) are the same, (C) A subsystem of two longitudinal tubulin dimers where the receptor is tubulin <i>k</i> and the ligand is its periodic image, where <i>k</i> runs from 1 to 13. Total number of residues may differ slightly between the GDP and GTP models.</p