Received: 10 February 2023 | Revised: 23 April 2023 | Accepted: 21 May 2023

DOI: 10.1002/cbin.12052

Cell Biology
RESEARCH ARTICLE International

In silico induced effect of N‐ε‐lysine acetylation

on microtubule stability and subsequent interaction
of microtubule‐associated proteins

Alexey Rayevsky1,2 | Elijah Bulgakov1,2 | Mohsen Sharifi3 |

Dariya Samofalova1,4 | Daniil Ozheredov5 | Pavel Karpov1 | Sergio Pantano6 |
1
Yaroslav Blume
1
Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, Кyiv, Ukraine
2
Department of Molecular Modeling, Enamine Ltd., Kyiv, Ukraine
3
RockGen Therapeutics, Little Rock, Arkansas, USA
4
R&D Department, Life Chemicals Inc., Niagara‐on‐the‐Lake, Ontario, Canada
5
Institute of High Technologies, Glushkova Ave, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
6
Institut Pasteur de Montevideo, Montevideo, Uruguay

Correspondence
Alexey Rayevsky, Institute of Food Abstract
Biotechnology and Genomics, National
Plant systems have been considered valuable models for addressing fundamental
Academy of Sciences of Ukraine, Оsypovskoho
str., 2A, Кyiv 04123, Ukraine. questions of microtubule (MT) organization due to their considerable practical utility.
Email: rayevsky85@gmail.com
Protein acetylation is a very common protein modification, and therate of

Present address
Mohsen Sharifi, Corteva Agriscience,
9330 Zionsville Road, Indianapolis,
Indiana 46268, USA.
Funding information
National Research Foundation of Ukraine,
Grant/Award Number: 0120U104883;
MERCOSUR Structural Convergence Fund,
Grant/Award Number: COF 03/11
acetylation can be modulated in cells in different biological states, and these
changes can be detected at a molecular level. Here, we focused on K40, K112, and
K394 residues as putative acetylation sites, which were shown to exist in both plants
and mammals. Such residual effect of acetylation causes critical but unclear effect on
MT stability. In turn, it was shown that acetylation indirectly affects the probability
of interaction with different MAPs (Microtubule‐associated proteins). In a multiscale
study using an all‐atom force field to reproduce several lattice‐forming elements
found on the surface the microtubule, we assembled a fragment of a plant
microtubule composed of nine tubulins and used it as a model object along with the
existing human complex. Triplets of tubulins assembled in a lattice cell were then
simulated for both human and plant protein complexes, using a coarse‐grained force
field. We then analyzed the trajectories and identified some critical deformations of
the MAP interaction surface. The initial coordinates were used to investigate the
structural scenario in which autophagy‐related protein 8 (ATG8) was able to interact
with the MT fragment.

KEYWORDS
acetylation sites, autophagy‐related protein, CHARMM36, Gromacs, SIRAH 2.0, tubulin

Cell Biol Int. 2023;1–11. wileyonlinelibrary.com/journal/cbin © 2023 International Federation for Cell Biology. | 1
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1 | INTRODUCTION
Microtubules (MT) are dynamic complex substructures. Having both
flexibility and strength, MT are vital for the functioning of
cytoskeleton network (Gudimchuk & McIntosh, 2021). In general,
MT can be considered as an assembly of structural (α and β
monomers) or functional (its heterodimers) elements (Figure 1). Many
proteins (MAPs, enzymes, etc.) were shown to interact differently
with these elements, and these interactions depended on the
protein's involvement in cell cycle‐related processes. Despite inten-
sive past and current research on plant microtubules and structural
and functional data, the mechanisms underlying MT reversible
stabilization and destabilization are not yet clear. The dynamics of
such an important structure is modulated by the number of side chain
modifications on the surface of the tubulin proteins and reversible
reactions of phosphorylation and acetylation (Gardiner & Marc, 2011;
Samofalova et al., 2019; Wloga et al., 2017). Posttranslational
modifications on the surface or at the interface of Protein–Protein
Interaction (PPI) s of tubulins can affect the balance between the
above processes, changing the probability of MAP association with
microtubules (Eshun‐Wilson et al., 2019). These data suggest, that
some changes on the surface or at the interface of PPI s are
associated with posttranslational modifications. Due to their high
therapeutic potential, MTs have been widely studied at different
levels in animal systems (Čermák et al., 2020; Liu et al., 2022;
Marchisella et al., 2016). Besides, plant systems are also considered
valuable models for characterizing fundamental issues of MT
organization and MAP interaction due to the considerable practical
F I G U R E 1 Structural representation of tubulin complexes. The predicted and experimentally confirmed acetylation sites on the surface of
Arabidopsis thaliana (a) and human (b) tubulins are illustrated. α‐tubulins and β‐tubulins are colored blue and green, respectively, and selected
acetylation sites are marked in red. (c) Schematic structure of a microtubule with structural intermediates in microtubule assembly and the two
most interesting acetylation sites, K40, and K394.
RAYEVSKY ET AL.
convenience, despite some differences between the two systems
(Rayevsky et al., 2019). This is especially interesting for studying the
indirect effect on PPI of some “house‐keeping” proteins
(Monastyrska et al., 2009).
For example, several studies have implicated the protein α‐
tubulin acetyltransferase (αTAT), which belongs to the class of
acetyltransferases and it is common to animal cells (Ilan, 2019).
Neither αTAT homologues nor other partners with the same putative
function were found in plants. However, HDAC6/12, MYST‐, and
p300‐like proteins, that ensure the reverse process are present in
plant cells (Adamakis et al., 2019; Raevsky et al., 2016). As recent
works have shown, some lysine residues on the surface of α‐tubulins
can be acetylated in both soluble and polymerized forms (dimers in
the microtubule apparatus), causing the development of autophagy
(Coleman et al., 2021; Lytvyn et al., 2018; Olenieva et al., 2019). At
the same time, the modified state is maintained due to the
deactivation of deacetylase enzymes. Following the possible ex-
planations presented in previous studies of the effects of α‐tubulin
acetylation on microtubule stability, we decided to investigate some
motor changes of MTs and interpret the available structural and
biochemical data (Eshun‐Wilson et al., 2019). Chemical replacement
of the terminal nitrogen in the of Lys structure by an acetate group is
one of the proven cases of widespread posttranslational modification
in α‐tubulin (Janke & Montagnac, 2017). The impact of most of the
putative acetylation sites has not yet been investigated experimen-
tally (Liu, Xiong, Ren, et al., 2015). To better understand the process
of structural changes (microtubule reorganization) and the subse-
quent interaction of microtubules with phagosomal elements, we
RAYEVSKY ET AL.
decided to simulate this effect of acetylation using in silico methods.
With this in mind, we chose three Lysine positions, that form a
triangle on a sagittal plane of the α‐tubulin molecule.
The results of our previous study made it possible to push these
findings further by creating a large model using the simultaneous
presence of several lattice‐forming elements found in the micro-
tubule, forming a lattice of proteins (Rayevsky et al., 2019). Wloga
et al. have already predicted and investigated the acetylation sites on
the surface of microtubules (K40, K112, and K394) (Wloga
et al., 2017). We performed a multiscale study using all‐atom force
field to recreate several of these locations. To do this, we assembled
a fragment of a plant microtubule, consisting of nine tubulins, and
used it as a model object together with the existing human complex
(Rayevsky et al., 2019; Vemu et al., 2016). Then human–plant protein
complexes, triplets of tubulins assembled in a lattice cell, were
modeled for 1 µs using a coarse‐grained (CG) force field. Clear
deformations of the MAP interaction surface, as well as the
intermolecular contacts, were analyzed. The initial coordinates were
used to investigate the structural scenario in which autophagy‐
related protein 8 (ATG8) was able to interact with the MT fragment.
2 | METHODS AND MATERIALS
Human and plant tubulin alpha‐4A chains (P68366 and Q0WV25)
tubulin beta‐4 chains (P04350 and P24636) were taken from the
UniProt database (http://www.uniprot.org) and structural templates
from the RCSB protein database (www.rcsb.org). The structures of
the Arabidopsis tubulins and ATG8 protein family have already been
generated and optimized (Rayevsky et al., 2019, Rayevsky,
Ozheredov, et al., 2021) The structures of Tubulin from Arabidopsis
thaliana have already been modeled based on the existing crystal
structures from Bos taurus and Sus scrofa (PDBID: 1Z2B, 1TUB) due
to the high degree of homology of 83%. The most satisfactory
templates for modeling ATG8 belong to homologous protein
structures from Solanum tuberosum (PDBID: 5L83).
The last 10 residues of the C‐terminal tail of tubulins, which
are normally very flexible and absent from X‐ray structures, were
reconstructed to provide a complete representation of the
SCHEME 1 Investigation protocol and computational tools are used to investigate the effect of acetylation.
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quaternary environment but were not included in any analysis
(Freedman et al., 2011).
The original residue topology data set, which is included in the
CHARMM36 force field package, has been modified by incorporating
additional information from the literature and the official CHARMM
website (Grauffel et al., 2010; Huang & MacKerell, 2013; Lee
et al., 2016). We then, used the full‐atom force field output as input
coordinates to increase the conformational sampling using the SIRAH
force field for CG simulations (Machado & Pantano, 2016; Machado
et al., 2019). CG parameters for acetylated lysine residues (AcKs)
were taken from Garay et al. (2020) The effects of acetylation were
studied based on a molecular dynamics (MD) simulation approach
using Gromacs 2019 software package. An assembled microtubule
lattice (three connected plant β‐α‐β tubulin trimers), with bound GTP
and GDP molecules was generated based on a similarly organized
complex [PDBID: 5JCO] and relaxed during 10 ns in the full‐atom
CHARMM36 force field and then simulated at the CG level for 1 µs
(Rayevsky, Ozheredov, et al., 2021) (Scheme 1).
The multiprotein complex was solvated in clear CG water
molecules within a computational box extending 30 Å from the
protein molecules. All parameter files for subsequent energy
minimization, stepwise constraint equilibration, and final production
MD were developed for GROMACS and required no modification.
The final “backmapping” procedure (reverse conversion of CG
“beads” to atoms), tailored to obtain minimized full‐atom models
was performed using a combination of VMD 1.9.3 (generation of a
protein structure file) and AmberTools 18 (minimization procedure)
software (Humphrey et al., 1996; Machado & Pantano, 2016). A
complete list of all simulated systems is presented in Table 1.
Protein structure stability and three‐dimensional deformation
were processed and analyzed with GROMACS build‐in tools and
visualized using PyMol 1.5 and VMD 1.9.3. Analysis of protein
complexes included a series of standard methods for comparison—
Root Mean Square Fluctuation (RMSF), Rg (Radius of gyration),
calculation of the PPI partition area, and conformational clustering of
the MD trajectory. The study of segments and individual areas of the
surface was performed by indexing the atom numbers of the
corresponding amino acid residues. The most stable sites, considered
to be the most likely interface for MAP binding, were obtained by
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T A B L E 1 Detailed description of conducted MD simulations in Arabidopsis thaliana and Homo sapiens using three different force field
parameter sets.
Simulation model (runs) Organism
Single α‐tubulin (wt + 3 Ac sites) A. thaliana
12‐meric lattice βαβα (wt) H. sapiens
12‐meric lattice βαβα (wt) H. sapiens
9‐meric lattice αβα/βαβ (wt) H. sapiens
9‐meric lattice αβα/βαβ (wt) H. sapiens
9‐meric lattice βαβ (3 Ac sites) A. thaliana
9‐meric lattice βαβ (3 Ac sites) H. sapiens
Abbreviation: MD, molecular dynamics.
clustering the RMSD values of the indexed atoms throughout the
second half of the MD simulation, which corresponded to the relaxed
state. Visualization of PPI interaction was presented using Python
scripts and calculated with PyMol tools.
We performed all PPI docking calculations using a local
installation of the HADDOCK 2.4 program, which also supports CG
docking (van Zundert et al. 2015, 2016). Docking was performed on
the based on Ambiguous Interaction Restraints (AIR) obtained from
constrained complexes (true interface). For this, all available solvent
residues, that could interact with ATG8 (Lys112, Tyr108, Gly410,
Glu411, Ser193, Glu155, Met413, Glu415, and Glu417) were
selected with at least one heavy atom within 3.9 Å of any heavy
atom of the partner molecule (Rayevsky, Ozheredov, et al., 2021).
The sampling parameters were stored at default in HADDOCK: 1000
models were generated for rigid body energy minimization (it0), 200
for the semiflexible segment refinement (it1), followed by 200 water
refinement steps (itw).
For effective use of existing computing resources and services in
calculations of molecular dynamics, we used our virtual laboratory
CSLabGrid, (Karpov, 2015; Rayevsky et al., 2022) which is part of the
Ukrainian National Grid (http://uran.ua/).
3 | RESULTS
In this study, we attempted to characterize the structural and
dynamic properties of MTs formed by acetylation of selected lysine
residues on the surface of α‐tubulins. For this purpose, we compared
both the structural changes in the individual elements of the system
and the interactions of the whole ensemble of molecules. We used
the structure of the human body as a structural model and evaluated
the effects of hyperacetylation on both systems on mammalian and
plant systems. Lysines at positions 40, 112, and 394 were selected
because of their spatial arrangement on the surface of α‐tubulin
(Figure 1).
Access to the K40 position is sterically prevented, as it is located
on the inner surface of the microtubule plate (Figure 1c) (Gaertig &
Wloga, 2012). This means t that it is not evident when the acetylation
RAYEVSKY ET AL.
Force field Number of particles Time (ns)
Charmm36 51,653 500
Charmm36 1,135,736 100
SIRAH 248,102 2000
Charmm36 921,293/905,253 100
SIRAH 248,098/155,311 1000
SIRAH 148,346 500
SIRAH 155,308 500
process takes place and how this posttranslational modification
affects the distal MAP binding sites. However, it was observed that
the MT assembly was significantly slowed when K40 was acetylated
in isolated tubulins (Portran et al., 2017). In contrast, the K112 and
K394 acetylation sites, face the outer side of the MT and can directly
influence MAP association. Recently, the role of K40 and K394 it has
been shown to be critical for the process of cytoskeleton
reorganization in both human and plant organisms (Liu, Xiong, Li,
et al., 2015). Furthermore, acetylation can affect the rigidity of
microtubule architecture and increase longitudinal contacts between
tubulins (Schaedel et al., 2015). These changes affect the binding
mode of many MAP proteins that interact with the microtubule
surface (Littauer et al., 1986).
3.1 | Modeling of all‐atom acetylated states
of plant α‐tubulin
All three positions were shown to be acetylated both in a soluble
state and in the composition of MT. Thus, we started three molecular
dynamics simulations of the α‐tubulin monomer acetylated at each
position. Full details on the conditions and methods for comparative
analysis of the trajectories are disclosed in a recent paper on the
effects of N‐acetylated K40 (Rayevsky, Ozheredov, et al., 2021).
Here, we demonstrate the results of MD simulations conducted on
unacetylated α‐tubulin and α‐tubulin with N‐acetylated K40, K112,
and K394 (Figure 2). The overall state of the protein depends
significantly on the degree of acetylation of K40 and K394.
Interestingly, after acetylation, all lysine residues changed their
original conformation, which was exposed to the solvent‐ to interact
with the protein surface. For example, AcK40 forms H‐bonds with
Q358 and H28 or another pair, D245 and E27, reducing the flexibility
of the surrounding region. Notably, K40 and K394 residues are
located near the interdimer interface, and their acetylation enhances
the longitudinal, but not the lateral, bonds s between α‐ and β‐tubulin
molecules (Eshun‐Wilson et al., 2019; Portran et al., 2017).
Following the RMSF plots, we observed that α‐tubulin acetyla-
tion at position 112 leads to the least effect on the monomer
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F I G U R E 2 Graph representationn of the structural changes in soluble α‐tubulin molecules of Arabidopsis thaliana caused by acetylation at
positions K40 (orange), K112 (green), and K394 (cyan) (a, b, and c, respectively). Residue‐oriented RMSF plots of acetylated proteins are
compared with those obtained from the trajectory of deacetylated tubulin. Colored dots indicate those residues, which form the corresponding
PPI interfaces to reflect their flexibility along the MD. The listed residues are those involved in the transient PPI interactions of each α‐tubulin in
human MT. MD, molecular dynamics; MT, microtubule.

structure. Notably, this modified amino acid is exposed to the
solvent and not involved in any obvious intermolecular interaction
in MT. In the context of MT, it is more likely that acetylation is
either sterically unfavorable or that the modification has no effect
on the already assembled MT. It is possible that the acetylated
soluble dimer or monomer could simply be excluded from the
polymerization process (Wloga et al., 2017). Our study showed that
acetylation at the K112 position had no critical effect on the protein
conformation, so this position was excluded from the subsequent
stage of MD simulations.
3.2 | CG simulation of an acetylated MT fragment
from A. thaliana
In an attempt to understand the role of acetylation in microtubule
reorganization and to visualize some of the structural changes, we
modeled a fragment of MT acetylated at Lysine residues 40 and 394.
Conducting research on the microtubule ring structure (even one coil
of MT from PDBID: 6CVN contains 14 dimers) is very challenging in
terms of computational demands and technical complexity due to the
large size of the system (Sinelnikova & Spoel, 2021). However,
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modern CG force fields can provide residual‐level accuracies
comparable to experimental methods, thus providing a convenient
alternative for modeling large‐scale systems (Darré et al., 2015;
Machado & Pantano, 2016; Machado et al., 2019). To expand the
dynamic sampling, we performed CG simulations starting from two
configurations derived from the aforementioned human complex,
including a 12‐mer peptide formed by αβαβ layers (1,135,736 atoms)
and also a 9‐mer peptide formed by βαβ layers (905,253 atoms). The
first system remained stable up to 5 μs, and changes in the fragment
of the microtubule structure were insignificant, almost not affecting
the extreme row of α‐tubulins (Supporting Information: Figure 1).
However, the model successfully visualized the process, as reported
in a recent study, demonstrating that SIRAH 2.0 is able to show some
weakening of the intermolecular contacts and bending of protofila-
ments (Supporting Information: Figure 2) (Fedorov et al., 2019;
Manka & Moores, 2018). Instead, the second one maintained
sufficient stability during the first 500 ns of the dynamics. However,
possibly due to the finite size effects, significant distortions in the
quaternary structure (weakening of intermolecular interactions) were
observed after 600 ns, causing separation of the αβ dimers from the
previous row. Ultimately, we decided to examine the effect of
acetylation on the first model.
2+
Using GTP/GDP molecules and Mg ions as cofactors we tried
to reproduce the most realistic scenario of microtubule degradation.
Normally, after incorporation of the α‐β dimer into linear protofila-
ments, the GTP molecule bound to the β‐tubulin binding site is
gradually hydrolyzed into GDP (Howard & Hyman, 2003). Numerous
events of GTP degradation led to a decrease in the stability of dimers
and subsequent disruption of MT.
In addition, molecular dynamics of the ensemble of plant
tubulins with AcK in all α‐tubulins revealed changes in inter-
molecular interactions. We analyzed the most constant period of
MD, namely, the last 300–400 ns of the simulation, when the
entire system is balanced and the probability of random structural
changes is the lowest, but the trends of global transitions are
already in sight. The most vivid illustration of the change in the
shape of the model could be the centroids of large conformational
clusters (Figure 3). Interestingly, the set of contacts in the complex
was observed to increase in the longitudinal direction upon
acetylation at position 40. In the case of AcK40, the mobility of
the loop is reduced, and the α‐tubulin molecule is able to form
more contacts with the bottom row of β‐tubulins. In the
deacetylated state, we observed loss of PPI contacts between
the α/β‐tubulin dimers and dissociation of the GDP molecules that
were located on β‐tubulin subunits.
In an attempt to clarify the mechanistic effects of acetylation on
the structure of tubulins, we analyzed the radius of gyration
(Supporting Information: Figure 3) and mobility of amino acid
residues. In particular, we paid attention to surface changes in the
total number of intermolecular contacts. It has been shown that the
compaction of the protein fold is inherent in the deacetylated
protein. For example, from analysis of the radius of Gyration the
central α‐tubulin, we concluded that acetylated tubulin is more
compact than its deacetylated form, which can demonstrate higher

F I G U R E 3 The intermolecular contacts and grids illustrate the location of the plant subunits in the initial state of the complex (a), as well as
the conformations of the modified complexes of the deacetylated system (b), K40ac (c), and K394ac (d), obtained after 400 ns of MD. The table
(top right) lists the calculated PPI (calories per mole per square angstrom) cross‐sectional areas. Graphical representation of structural changes in
central, deacetylated plant α‐tubulin and molecules, modified at positions K40 (orange) and K394 (cyan) exposure with the less “edge effect.”
MD, molecular dynamics.
RAYEVSKY ET AL.
flexibility in external secondary structures. This is consistent with
reports indicating that acetylation reduces both the microtubule
nucleation and polymerization rates (Portran et al., 2017). As a result,
we observed a deformation of the initial geometry, namely,
unmodified dimers partially lost contact with the lower row of β‐
tubulins. In turn, the entire structure of this β‐tubulin chain also
weakened. In the cases of α‐tubulin acetylation, the contacts
between proteins in the complexes remained stable, and the
longitudinal bundles of β‐α‐β tubulins shifted towards each other,
changing the angle of the helix. It is known that increasing the level of
acetylation must somehow stabilize the MT ensemble, and these data
are consistent with the effect of K40 and K394 modifications on the
structural rearrangement during MD simulations.
During these MD simulations, the GTP molecules did not
change their location, while the GDP molecules were released
from the deacetylated complex (Supporting Information:
Figure 4). Moreover, this occurred in both rows of β‐tubulins as
a result of some decrease in the areas of PPI interaction between
dimers in the longitudinal and transverse directions. Meanwhile,
the acetylated complexes did not show significant changes in the
location of the substrates.
3.3 | CG simulation of a MT fragment from
H. sapiens
We also conducted similar simulations on the human complex to
reveal some trends in the behavior of these two acetylation modes
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(Figure 4). In addition, animal complexes are well studied and
supported with biochemical and crystallographic data. The compari-
son of protein–protein surface and putative binding sites provides
valuable insights into yet‐unexplored site similarities. Various stages
of computational and biochemical research can benefit from this
knowledge. Based on the MD results, we concluded that the
sequence of events in MT regulation should be the same as we
observed in the plant system. First of all, the variation of the shape
and size of each α‐tubulin depends on the degree of acetylation and
subsequent disruption of contacts in both vertical and horizontal
directions. We, then found that the human system showed an even
more remarkable effect of acetylation, than the plant system. Here
we refer to the tables containing the values of maintained contacts
between neighboring subunits in the case of its acetylated state.
3.4 | Structural prerequisites leading to the
formation of the microtubule/ATG8 complex
We were interested in the effect of hyperacetylation not only on the
stability and dynamics of the microtubule itself, but also on the ability
to associate MAP proteins. It was shown that acetylated micro-
tubules are required for the fusion of autophagosomes with
lysosomes and the regulation of the autophagy process (Bánréti
et al., 2013; Xie et al., 2010). In this study, we focused our attention
on the binding site of motor protein kinesins (which mediates
movement away from the nucleus) and LC3/ATG8 which are
involved in the maturation and trafficking cascade of of mature

F I G U R E 4 Calculated PPI interface areas for different acetylation modes of human MT fragment. General surface representations (α‐tubulin
shown in green) and visualization of intermolecular contacts for deacetylated, K40ac, and K394ac modified complexes, obtained after 400 ns of
MD (a–d, respectively). Graphical representation of the structural changes in central, the less “edge effect” exposure, deacetylated plant
α‐tubulin and molecules modified at positions K40 (orange) and K394 (cyan) (d). MD, molecular dynamics.
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autophagosomes. In an attempt to achieve correlation with other
biochemical data, we derived structural models of tubulins in complex
with ATG8 to investigate the dependence of the PPI map on changes
in α‐tubulin state of acetylation.
To investigate the effect of acetylation on the MT‐MAP
association we prepared our protein–protein docking models using
the results described in the previous paragraphs. We carefully tested
the binding of LC3 (the homologue of ATG8 in human cells) on the
tubulin surface using key residues from the literature (Monastyrska
et al., 2009). We previously reported the results of our study on the
analysis of reconstructed (plant/animal) MT fragments and surface
probing (Rayevsky et al., 2019, Rayevsky, Ozheredov, et al., 2021).
From this, we planned to determine mechanistic prerequisites for the
structural changes of the entire MT (Rayevsky, Samofalova,
et al., 2021).
A set of terminal arginine residues from ATG8, similar to those
crucial amino acids from LC3, was used to constrain the AIRs
restraints in the Haddock PPI model. In turn, based on our previous
studies, the C‐terminal residues were selected as an epitope tag for
ATG8 binding to the ensemble from A. thaliana (Rayevsky,
Ozheredov, et al., 2021). The PPI docking results showed an exact
match of the surface profiles for the acetylated systems, as recently
shown for α‐tubulin acetyltransferase (Figure 5) (Atherton et al., 2020;
Leverett et al., 2016; Rayevsky et al., 2019). In the case of a
deformed deacetylated system, the docking initial complexes did not
show a sufficiently close location of the ATG8 molecule on the MT
surface.
The initial docking positions of ATG8 confirmed the obvious
stabilizing effect of MT acetylation at K40 and K394 positions on the
probability of the ATG8‐MT association process. We identified a set

F I G U R E 5 Spatial superposition of the human microtubule fragments co‐crystalized in complex with a set of positively charged MAPs in the
region of the negatively charged tubulin's C‐terminal (PDBID: 5KX3, 6ZPI, 3JAK). Here we represent a Tubulin tyrosine ligase (a), Kinesin‐like
protein (b) and EB3 proteins (c). Similarly, ATG8 molecule (magenta) tightly interacts with a relaxed fragment of plant MT containing K40 and
K394 acetylated residues of α‐tubulin (d and g, respectively). Frontal and vertical view of the initial state (e, h) and final, relaxed (f, i) MT fragment
with deacetylated α‐tubulins. MD, molecular dynamics; MT, microtubule.
RAYEVSKY ET AL.
of prerequisites for a more favorable interaction between ATG8 and
the acetylated system compared to the deacetylated one, and
demonstrate this in Figure 5. It is clear that the β‐tubulin↓ row is
stable and compact in the initial state of the deacetylated system
(Figure 5e) and after MD simulation rapidly loses all native contact
with the upper tubulin dimer (Figure 5f). Finally, this causes an
imbalance of the entire interaction network, shown from both the
frontal and lateral sides. In turn, this forms another prerequisite—the
central α2‐tubulin (Figure 5h,i) is pressed into the plane of the MT
fragment, thus preventing its association with ATG8.
4 | C ONC LUS I ON S
Based on the results of in silico modeling, it is reasonable to suggest
that the mechanism of structural changes that appears to cause the
destruction or stabilization of MT depends on the level of
posttranslational modification. MD simulations of a single protein
showed that acetylation has a notable effect on surface stiffness.
Interestingly, acetylation of K112 does not critically affect the overall
structure of tubulin, so it was excluded from our study. At this stage
we demonstrated the ability of α‐tubulin to maintain its shape and
surface under the influence of acetylation. We studied the most
widely‐spread acetylated monomeric forms (positions K40, K112,
and K394) and analyzed the effect on the α‐tubulin interfaces in A.
thaliana. Further analysis demonstrated structural differences
between nonacetylated and acetylated states of the MT lattice
simulated at the CG level, confirming that the expected trends in
acetylation‐dependent stabilization for K40 and K394 thus altered.
Nonacetylated lysine is less flexible compared to the acetylated
form and it is constantly oriented into the protein body. In turn, the
acetylated form of lysine establishes H‐bonds with the surrounding
amino acids and almost always adheres to the surface. Together with
the transformation of charge on the surface, the orientation of lysine
prevents critical structural deformations and facilitates the interaction
process. We also were able to image some experimental observations,
suggesting the predominance of longitudinal contacts versus transverse
contacts. We observed that the addition of an acetyl group to K40 or
K394 reduces some of the motion of the surrounding regions, providing
stabilization of the longitudinal bonds, while the overall size fluctuation
of the entire structure is somewhat increased. Thus, we have
demonstrated a cascade of events associated to the acetylation of
certain residues on α‐tubulin which leads to the stabilization of the MT
polymeric structure. In addition, we found that ATG8 preferably binds
the acetylated form of α‐tubulin, as a part of the MT, due to the
deformations of the outer surface of polychain complex. Our model was
mostly designed to show significant internal deformation during the
dynamics. Finally, the original reason of the observed changes is in the
different level of the α‐tubulin compactness, so we are going to apply
the model to investigate an extended panel of modifications, detected
by means of proteomics methods. At the same time, these validated
systems of MT fragment could be a useful tool for our future studies on
ATG8 acetylation and lipidation, allowing us to examine the impact of
Cell Biology | 9
International
these posttranslational modifications on the dynamics of autophago-
some formation and as a key to delve into the details of serine/
threonine kinase (AuTophaGy related protein 1) regulation and its
complex functioning.
A UT H O R C O N T R I B U TI O NS
Alexey Rayevsky: Conceptualization; data curation; investigation;
methodology; writing—original draft. Elijah Bulgakov: Investigation;
writing—review & editing. Mohsen Sharifi: Resources; software;
visualization; writing—review & editing. Dariya Samofalova: Formal
analysis; funding acquisition; investigation. Daniil Ozheredov: Valida-
tion; visualization; writing—review & editing. Pavel Karpov: Formal
analysis; funding acquisition; resources. Sergio Pantano: Resources;
software; supervision; writing—review & editing. Yaroslav Blume:
Project administration; supervision; writing—review & editing.
ACKNOWLEDGME NT S
The work was supported by the National Research Foundation of Ukraine
(https://nrfu.org.ua/) under the project “Enzymes of posttranslational
modifications of microtubules proteins, as targets for excitability inhibition
of primary nociceptive neurons of peripheral nervous system,” State
Registration № 0120U104883, and using the capacities of the Grid
Cluster of the Institute of Food Biotechnology and Genomics of the
National Academy of Sciences of Ukraine and the virtual organization
CSLabGrid (http://ifbg.org.ua/en/cslabgrid). This work was partially
supported by FOCEM (MERCOSUR Structural Convergence Fund),
COF 03/11. The acknowledgment is also given to Dr Atena Farhangian
at the University of Vermont and Keaton Rath at Indiana
University–Purdue University Indianapolis for the helpful comments on
the manuscript. Likewise, authors are grateful to Mr. Vladimir Rayevsky
for the valuable assistance with graph visualizations of the manuscript.
CONFLIC T OF INTEREST STATEM ENT
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
Research data are not shared.
ORC I D
Alexey Rayevsky http://orcid.org/0000-0002-7596-6294
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SUPP ORTING INFO RM ATION
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
How to cite this article: Rayevsky, A., Bulgakov, E., Sharifi, M.,
Samofalova, D., Ozheredov, D., Karpov, P., Pantano, S., &
Blume, Y. (2023). In silico induced effect of N‐ε‐lysine
acetylation on microtubule stability and subsequent
interaction of microtubule‐associated proteins. Cell Biology
International, 1–11. https://doi.org/10.1002/cbin.12052