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. 2020 Jun 16;118(12):2938-2951.
doi: 10.1016/j.bpj.2020.04.028. Epub 2020 May 1.

Microtubule Simulations Provide Insight into the Molecular Mechanism Underlying Dynamic Instability

Affiliations

Microtubule Simulations Provide Insight into the Molecular Mechanism Underlying Dynamic Instability

Dudu Tong et al. Biophys J. .

Abstract

The dynamic instability of microtubules (MTs), which refers to their ability to switch between polymerization and depolymerization states, is crucial for their function. It has been proposed that the growing MT ends are protected by a "GTP cap" that consists of GTP-bound tubulin dimers. When the speed of GTP hydrolysis is faster than dimer recruitment, the loss of this GTP cap will lead the MT to undergo rapid disassembly. However, the underlying atomistic mechanistic details of the dynamic instability remains unclear. In this study, we have performed long-time atomistic molecular dynamics simulations (1 μs for each system) for MT patches as well as a short segment of a closed MT in both GTP- and GDP-bound states. Our results confirmed that MTs in the GDP state generally have weaker lateral interactions between neighboring protofilaments (PFs) and less cooperative outward bending conformational change, where the difference between bending angles of neighboring PFs tends to be larger compared with GTP ones. As a result, when the GDP state tubulin dimer is exposed at the growing MT end, these factors will be more likely to cause the MT to undergo rapid disassembly. We also compared simulation results between the special MT seam region and the remaining material and found that the lateral interactions between MT PFs at the seam region were comparatively much weaker. This finding is consistent with the experimental suggestion that the seam region tends to separate during the disassembly process of an MT.

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Figures

Figure 1
Figure 1
(a) Schematic view of the MT structure. Each tubulin domain contains around 440 residues, which is represented by one bead. The three PFs included in the red rectangle consist of our first MT patch simulation system, whereas all the 13 PFs with a length of 16 tubulin domains are included in our closed MT segment simulation system. (b) Shown is a cartoon representation of the αβ tubulin heterodimer, which serves as the basic building block of the MT. The nonexchangeable nucleotide binding site (N-site), located at the intradimer interface, can only bind GTP molecules. In contrast, the E-site, exposed at the top of β tubulin domain, can bind either GTP or GDP molecules. (c) Illustration of MT dynamic instability is shown. MT can switch between the growth state and shrinkage state, depending on the presence of GTP cap. To see this figure in color, go online.
Figure 2
Figure 2
Evolution of PF lengths in lattice patch simulations starting from different initial structures: (a) EB3-bound, 13-PF starting structures 3JAL (GTP) and 3JAR (GDP) and (b) kinesin-bound, 14-PF starting structures 3JAT (GTP) and 3JAR (GDP). The initial lengths, before any equilibration, are labeled by large dots at t = 0. To see this figure in color, go online.
Figure 3
Figure 3
(ac) Comparison of bending angle distributions of the MT PFs in the GTP (purple), GDP (green), and GDP + Pi (cyan) states. (d) The probability distributions of bending angle differences between neighboring PFs in three different nucleotide states are given. To see this figure in color, go online.
Figure 4
Figure 4
Distributions of the tangential component of PF bending angle for GTP (purple), GDP (green), and GDP + Pi (cyan) state simulations. The middle PF of the MT patch is used for calculation in each case. To see this figure in color, go online.
Figure 5
Figure 5
Distributions of nonbonded interaction energy between lateral interacting αβ tubulin dimers at the top of MT PFs in GTP (purple), GDP (green), and GDP + Pi (cyan). The interaction strengths between PF1 and PF2 are shown in (a), while PF2 and PF3 are shown in (b). To see this figure in color, go online.
Figure 6
Figure 6
The bending angle distributions computed from 500 ns MT patch simulations using alternative initial structures PDB: 3JAT (GTP) and PDB: 3JAS (GDP). (ac) Comparison of bending angle distributions of the MT PFs in the GTP (purple) and GDP (green) states is shown. (d) The distribution of nonbonded interaction energy between lateral interacting αβ tubulin dimers at the top of MT PFs in GTP (purple) and GDP (green) states is shown. To see this figure in color, go online.
Figure 7
Figure 7
(a) Superimposition of the lateral interacting αβ tubulin dimers at the top of neighboring MT PFs for the GTP (green) and GDP (cyan) states. The M-loop, which is crucial for lateral interactions, is shown in yellow. (b and c) Shown is a magnified view of the lateral interactions formed between the M-loop and the other side for GTP (b) and GDP (c) states. Key residues K60, Q85, R88, P89, and Y283 are shown using “sticks” representation in PyMol. To see this figure in color, go online.
Figure 8
Figure 8
Residue contact map between lateral interacting β tubulin domains at the top of MT PF1 and PF2 calculated from (a) GTP state and (b) GDP state MT patch simulations, showing the loss of lateral contacts in GDP state simulation. To see this figure in color, go online.
Figure 9
Figure 9
(a) The average bending angle distributions of all the 13 PFs in closed MT segment simulations for the GTP (purple) and GDP (green) states. (b) Shown is the probability distribution of bending angle difference between neighboring PFs at the nonseam interface for the GTP (purple) and GDP (green) states. To see this figure in color, go online.
Figure 10
Figure 10
Length variations in the upper half of PF1 during a 200 ns simulation involving a closed MT segment simulation for both the GTP (purple) and GDP (green) states. The initial length measured in cryo-EM structures are labeled by spheres located at t = 0. To see this figure in color, go online.
Figure 11
Figure 11
The distribution of nonbonded interaction energy between lateral interacting αβ tubulin dimers at the top of the MT PFs. The distributions are distinguished by different nucleotide states (GTP or GDP) as well as according to the type of lateral interface (seam or non-seam). To see this figure in color, go online.
Figure 12
Figure 12
(a) Superimposition of lateral interacting tubulin domains at the α|β seam interface. The top figure shows the view from inside the MT lattice, whereas the bottom figure shows the view from outside the MT wall. The tubulin domains are colored in green for the GTP state and cyan for the GDP state. (b) Detailed structures around the M-loop lateral interacting interface are shown. The M-loop of the β tubulin domain is shaded yellow for the GTP state and orange for the GDP state, with the key residue Y283 shown as sticks for both states. (c) Shown are detailed structures around the lateral interacting interface between helix 3 in the α tubulin domain and the other side. Note that the structures in this panel are viewed from the outside of the MT wall, as in the bottom figure of (a). The helix 3 structure is shaded yellow for the GTP state and orange for the GDP state. Key interaction residue pairs found in the GTP state (but absent in the GDP state) are shown as sticks. To see this figure in color, go online.

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