Three-Dimensional Reconstruction of a Full-Size GABAB Receptor
The three-dimensional (3D) pattern of a full-size GABAB receptor has been reconstructed using computer techniques. To simulate a real microenvironment for the GABAB receptor, the latter was embedded in the bilipidic membrane with the corresponding salt-water environment. Since homology modeling o...
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Інститут фізіології ім. О.О. Богомольця НАН України
2015
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irk-123456789-1482152019-02-18T01:23:37Z Three-Dimensional Reconstruction of a Full-Size GABAB Receptor Nyporko, A.Yu. Naumenko, A.M. Golius, A. Tsymbaliuk, O.V. Shapoval, L.M. Davidovska, T.L. The three-dimensional (3D) pattern of a full-size GABAB receptor has been reconstructed using computer techniques. To simulate a real microenvironment for the GABAB receptor, the latter was embedded in the bilipidic membrane with the corresponding salt-water environment. Since homology modeling of the GABAB receptor is among the computational methods allowing one to predict 3D coordinates when experimental data are not available, we reconstructed the structure of a full-size GABAB receptor by stepwise homology modeling of individual subunit parts. The stability of receptor subunits was evaluated by calculating the molecular dynamics. It has been found that C-terminal domains of the intracellular receptor show a tendency toward compaction, and coiled-coil areas form a structure almost identical to that specified by crystallization of these fragments. The structure obtained can be applied for further examination of the structural mechanisms of GABAB receptor interaction with GABA agonists and antagonists. It is quite evident that molecular dynamics computations might be a valuable tool in probing details of the receptor function. Проведена реконструкція просторової структури повнорозмірного ГАМКВ-рецептора. Для імітації реального мікросередовища ГАМКВ-рецептор був вбудований у біліпідну мембрану з відповідним водно-сольовим мікрооточенням. Оскільки гомологічне моделювання ГАМКВ-рецептора є важливим обчислювальним методом прогнозування просторових координат, коли експериментальні дані щодо структури не є доступними, ми реконструювали структуру повнорозмірного ГАМКВ-рецептора з використанням ступінчастого гомологічного моделювання окремих частин субодиниць. Стабільність субодиниць рецептора оцінювали, розраховуючи молекулярну динаміку. Було показано, що субодиниця модельованого ГАМКВ-рецептора складається з позаклітинного, трансмембранного і внутрішньоклітинного доменів. Встановлено, що внутрішньоклітинні С-термінальні домени рецептора мають тенденцію до компактизації, а надспіралізовані ділянки утворюють структуру, майже ідентичну до тої, що зумовлена кристалізацією цих фрагментів. Проведена реконструкція просторової структури повнорозмірного рецептора ГАМКВ є адекватною і такою, що може бути корисною для подальшого дослідження структурних механізмів взаємодії даного рецептора з агоністами і антагоністами ГАМК. Моделювання молекулярної динаміки може бути важливим інструментом вивчення деталей структури і динаміки рецептора. 2015 Article Three-Dimensional Reconstruction of a Full-Size GABAB Receptor / A.Yu. Nyporko, A.M. Naumenko, A. Golius, O.V. Tsymbaliuk, L.M. Shapoval, T.L. Davidovska // Нейрофизиология. — 2015. — Т. 47, № 5. — С. 429-440. — Бібліогр.: 45 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148215 615.213:54.057: 547.79 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
The three-dimensional (3D) pattern of a full-size GABAB receptor has been reconstructed
using computer techniques. To simulate a real microenvironment for the GABAB receptor,
the latter was embedded in the bilipidic membrane with the corresponding salt-water
environment. Since homology modeling of the GABAB receptor is among the computational
methods allowing one to predict 3D coordinates when experimental data are not available, we
reconstructed the structure of a full-size GABAB receptor by stepwise homology modeling of
individual subunit parts. The stability of receptor subunits was evaluated by calculating the
molecular dynamics. It has been found that C-terminal domains of the intracellular receptor
show a tendency toward compaction, and coiled-coil areas form a structure almost identical
to that specified by crystallization of these fragments. The structure obtained can be applied
for further examination of the structural mechanisms of GABAB receptor interaction with
GABA agonists and antagonists. It is quite evident that molecular dynamics computations
might be a valuable tool in probing details of the receptor function. |
| format |
Article |
| author |
Nyporko, A.Yu. Naumenko, A.M. Golius, A. Tsymbaliuk, O.V. Shapoval, L.M. Davidovska, T.L. |
| spellingShingle |
Nyporko, A.Yu. Naumenko, A.M. Golius, A. Tsymbaliuk, O.V. Shapoval, L.M. Davidovska, T.L. Three-Dimensional Reconstruction of a Full-Size GABAB Receptor Нейрофизиология |
| author_facet |
Nyporko, A.Yu. Naumenko, A.M. Golius, A. Tsymbaliuk, O.V. Shapoval, L.M. Davidovska, T.L. |
| author_sort |
Nyporko, A.Yu. |
| title |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor |
| title_short |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor |
| title_full |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor |
| title_fullStr |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor |
| title_full_unstemmed |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor |
| title_sort |
three-dimensional reconstruction of a full-size gabab receptor |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2015 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/148215 |
| citation_txt |
Three-Dimensional Reconstruction of a Full-Size GABAB Receptor / A.Yu. Nyporko, A.M. Naumenko, A. Golius, O.V. Tsymbaliuk, L.M. Shapoval, T.L. Davidovska // Нейрофизиология. — 2015. — Т. 47, № 5. — С. 429-440. — Бібліогр.: 45 назв. — англ. |
| series |
Нейрофизиология |
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2025-07-12T18:38:23Z |
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| fulltext |
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 429
UDC 615.213:54.057: 547.79
A. Yu. NYPORKO,1 A. M. NAUMENKO,1 A. GOLIUS,2 O. V. TSYMBALIUK,1
L. M. SHAPOVAL,3 and T. L. DAVIDOVSKA1
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
Received May 25, 2014
The three-dimensional (3D) pattern of a full-size GABAB receptor has been reconstructed
using computer techniques. To simulate a real microenvironment for the GABAB receptor,
the latter was embedded in the bilipidic membrane with the corresponding salt-water
environment. Since homology modeling of the GABAB receptor is among the computational
methods allowing one to predict 3D coordinates when experimental data are not available, we
reconstructed the structure of a full-size GABAB receptor by stepwise homology modeling of
individual subunit parts. The stability of receptor subunits was evaluated by calculating the
molecular dynamics. It has been found that C-terminal domains of the intracellular receptor
show a tendency toward compaction, and coiled-coil areas form a structure almost identical
to that specified by crystallization of these fragments. The structure obtained can be applied
for further examination of the structural mechanisms of GABAB receptor interaction with
GABA agonists and antagonists. It is quite evident that molecular dynamics computations
might be a valuable tool in probing details of the receptor function.
Keywords: GABAB receptor, subunit composition, 3D structure, bilipidic membrane,
molecular dynamics.
1 Institute of High Technology, Taras Shevchenko National University, Kyiv,
Ukraine
2 Jackson State University, Jackson, Mississippi, USA.
3 Bogomolets Institute of Physiology, National Academy of Sciences of
Ukraine, Kyiv, Ukraine.
Correspondence should be addressed to
A. M. Naumenko (e-mail ganna.naumenko@gmail.com),
A. Golius (e-mail anastasia@icnanotox.org), or
L. M. Shapoval (e-mail shapoval@biph.kiev.ua).
INTRODUCTION
γ-Aminobutyric acid (GABA) is, probably, the most
important inhibitory neurotransmitter in the mam-
malian CNS. This transmitter is extensively distributed
in the brain and plays a crucial role in reducing the
neuronal excitability throughout the nervous system. It
has been reported [1] that 30-40% of all CNS neurons
utilize GABA as the primary neurotransmitter. Since
there are about 40% of all synapses in the brain working
with GABA and, therefore, having GABA receptors,
the latter are believed to be the most common in the
mammalian CNS.
The presence of two types of postsynaptic receptors
in the brain that recognize GABA, namely GABAA
and GABAB ones, has been well documented. GABAA
receptor-mediated tonic inhibition plays an important
role in the CNS functioning. These receptors, linked
directly to binding sites of ion channels, are located
in the cell membrane and contain two functional
domains, an extracellular one that binds the
neurotransmitter and a membrane-spanning domain
that forms an ion channel. Since GABAA receptors
combine transmitter-binding and channel functions
into a single molecular entity, they are also frequently
qualified as ligand-gated ion channels. It has been
reported that ionotropic GABAA receptors contribute
to an increase in the conductance for chloride ions. In
electrophysiological studies using voltage-clamp and
single-channel recording techniques, the operation
of a GABAA receptor-gated Cl– ion channel has been
described in detail [2, 3]. Activation of such a channel
results in hyperpolarization of the neuronal membrane,
and this increases the threshold for generation of an
action potential (AP) in the case of action of excitatory
transmitters that depolarize the membrane. Shunting
of the cell membrane (a drop in its resistance)
accompanying activation of GABA receptors also
decreases the excitability of postsynaptic neurons.
GABAA receptors are multimers formed by at
least four or five individual protein subunits. It is
believed that the subunit compositions of most GABAA
receptors in various brain regions and even in various
neurons within a given region may be dissimilar.
GABAB receptors were identified when it became
clear that GABA can potently inhibit depolarization-
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5430
A. Yu. NYPORKO, A. M. NAUMENKO, A. GOLIUS, et al.
induced neurotransmitter (norepinephrine) release in
brain slices, but a number of GABAA receptor agonists
were found to be unable to mimic GABA-induced
inhibition of such neurotransmitter release [4]. Now it
has become obvious that GABAB receptors are located
on both post- and pre-synaptic membranes, and these
receptors do not include ion channels as a part of their
structure. Instead, they affect channels by activation
of intermediate molecules called G proteins. GABAB
metabotropic receptors are also called G protein-
coupled ones. Although there is a considerable body of
evidence that a large proportion of GABAB receptors
are coupled to G proteins, it has been also reported
that some presynaptic GABAB receptors may be
directly linked to K+ channels, since activation of these
receptors in many brain regions results in an increase
in the K+ channel conductance, with a resultant
hyperpolarization of the neuronal membrane [2, 4].
In the postsynaptic membrane, GABAB receptors
trigger, through G proteins, a cascade of intracellular
reactions leading to the opening of potassium channels
in the postsynaptic membrane [5]. Due to this event,
inhibitory postsynaptic potentials (IPSPs) lasting
hundreds of milliseconds develop. According to
the respective IPSP kinetics, GABAB receptors are
easily distinguished from GABAA receptors [6]. It
has been also reported that GABAB receptors regulate
the function of extrasynaptic GABAA receptors via a
postsynaptic mechanism [7], mediate slow inhibitory
synaptic neurotransmission, and play a key role in
long-term synaptic plasticity [8, 9]. There is evidence
that GABAB receptors are involved in neuronal
migration and positioning [10, 11]. Disruption of
GABAB receptor-mediated synaptic pathways is
implicated in many diseases, including neuropathic
pain, spasticity, drug addictions, hyperalgesia,
memory disorders, muscle spasticity, schizophrenia,
and epilepsy [12-16].
The GABAB receptor is a heterodimer, with an
extracellular domain containing a neurotransmitter
binding site and an intracellular domain that binds to
G proteins. This receptor is composed of two subunits,
GABAB1 (R1) and GABAB2 (R2), which differ from
each other in their N-terminal amino acid sequences
and arise due to alternative splicing [17, 18]. Each
subunit consists of a large extracellular module
called Venus flytrap (VFT), seven transmembrane
domains, and an intracellular C-terminal domain.
GABAB1 and GABAB2 subunits demonstrate a 54%
similarity in their amino acid sequence, but only the
extracellular domain of GABAB R1 can bind ligands,
such as GABA, baclofen, and orthosteric antagonists
(CGP54626, CGP64213, etc.) [19–21]. It is believed
that the formation of fully functional GABAB receptors
requires co-assembling of both R1 and R2 subunits of
the GABAB receptor [17, 22-26]. In the membrane,
GABAB receptors bind to G protein composed of α,
β, and γ subunits [27]. This is G protein that ensues
interaction of GABAB receptors with presynaptic
voltage-gated N- and P/Q-type calcium channels
[28, 29]. Multiple isoforms of human GABAB R1
subunit (GABAB R1a, GABAB R1b, GABAB R1c, and
GABAB R1e) have been described, but only GABAB R2
has been adequately identified [30]. It should be noted
that, at present, the structural basis for interaction
between GABA and the GABAB receptor has still
not been elucidated. It is obvious that computational
methods for predicting the 3D coordinates can be
beneficial for such a biomedical research, as well for
homology modeling. This technique is applied in the
situations where experimental structural data are not
available but needed. Molecular dynamics simulation
has also become relevant in the studies of such
biological systems.
In our study, we reconstructed the 3D structure of a
full-size GABAB receptor in a real microenvironment
using computer-based techniques.
METHODS
The amino acid sequences of the human GABAB
receptor subunits were retrieved from the international
database UniProt (http://www.uniprot.org/ [32]; the
access number for GABABR1 is Q9UBS5, and that for
GABABR2 is O75899).
To start the operations with protein 3D structures,
we used the atomic coordinate data deposed in Protein
Data Bank (PDB, http://www.rcsb.org/pdb/home/
home.do) [33]. The respective data are based on
the results of X-ray crystallographic analysis of the
extracellular domain of the GABAB R2 subunit at a
0.238 nm resolution (R-factor, 0.202), which has been
stored in the 4F11 record. This record is not complete,
as amino acid residues 42-466 of the extracellular
domain of the GABAB R2 subunit are enclosed in
the crystal under study. Structural data have been
deciphered for the receptor fragment 52-466.
The results of X-ray crystallographic analysis of an
intracellular coiled-coil heterodimer of the GABAB
receptor at a 0.162 nm resolution (R-factor, 0.217)
are stored in the 4PAS record. In the crystal under
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 431
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
study, amino acid residues 884-918 of the GABAB R1
receptor subunit and residues 779-817 of the GABAB
R2 subunit are enclosed.
Since homology modeling of the GABAB receptor
is among the computational methods to predict 3D
coordinates when experimental structural data are not
available [34], we reconstructed the structure of a full-
size GABAB receptor by stepwise homology modeling
of individual subunit parts.
The choice of optimal fold templates was based
on the structure integrity, percentage of identity,
percentage of similarity, and qualitative criteria
of spatial models [35]. The quality of the modular
architecture of protein structures was estimated using
a web server MolProbity as a general-purpose web
service, which can calculate and display the H-bond
and van der Waals contacts in the interfaces between
components, offering qualitative validation for the
3D structures of proteins [36]. The modules were
integrated in the overall 3D structure using home-
made software FlexBones.
Optimization for geometric reconstruction of the
patterns was performed using Amber3 force field [37]
and a method of the conjugate gradient [34, 38, 39].
The 3D structure and character of protein styling of the
chain were analyzed using Swis-PdbViewer 1.9.1 [40].
Visualization and analysis of the contact surfaces and
potential dimerization interfaces were handled through
desktop-based DS Visualizer software, versions 2.0
and 3.5.
We used a SymmDock web-tool [41] for prediction
of spatial structure of complete receptor by
geometrically based molecular docking. To simulate
the real microenvironment for a GABAB receptor, the
latter was embedded in the bilipidic membrane with
the corresponding salt-water environment. The next
optimization of the receptor spatial structure was
performed with GROMACS software (version 4.5.3)
[42] using a Charmm27 force field [43, 44].
The stability of receptor subunits in complex
biological membranes was evaluated by calculating
the molecular dynamics using GROMACS software.
The results of calculation of such dynamics for a
GABAB receptor/biological membranes complex
were evaluated basing on the root-mean-square
deviations (RMSD) between atoms, the root-mean-
square fluctuations (RMSF), and energy of non-valent
interactions. Visualization of complex behavior during
the molecular dynamics was performed using Visual
Molecular Dynamics 1.6.1 software [45].
RESULTS AND DISCUSSION
Despite the evident functional significance of GABAA
and GABAB receptors, the question of structural
interactions between GABA and these receptors still
remains open. It is known that interaction of GABA
with GABAB receptors is provided by molecular
conformation of the latter. The position of nitrogen and
oxygen atoms, as well as the distance between these
atoms causes the formation of a transmitter-receptor
complex that alters the membrane conductivity due to
the formation of pores in the membrane.
A model of the GABAB receptor was built according
to amino acid sequences obtained from the RCSB.PDB
(Protein Data Bank) database using the specialized
program Deep View – TheSwissPdbViewerv3.7.
To build the 3D model of the GABAB structure,
we used a crystallized fragment of the extracellular
domain of the R2 subunit (amino acid residues 52-466)
[20]. The corresponding entry in the PDB database
has a number 4F11 (Fig. 1). This structure shows a
high quality according to the MolProbity criteria. A
coiled-coil heterodimer of the intracellular domain of
the GABAB receptor containing 884-918 amino acid
residues of subunit R1 and 779-817 residues of subunit
R2 [31] has an appropriate entry in the PDB database
(number 4PAS) (Fig. 1.2).
Correct prediction of the protein structure by the
amino acid sequence may be achieved in two ways,
namely superposition of the known spatial structure of
the homologous protein and a method of “threading”
with step-by-step addition of short fragments and
iterative optimization of the energy systems. Since
the GABAB receptor does not have homologs with the
fully deciphered spatial structure, we reconstructed
the latter for the GABAB R1 and R2 subunits by
stepwise modeling of some homologous parts of these
subunits using Robetta web-based tools. To perform
these operations, the amino acid sequence of the each
F i g. 1. Structure of a GABAB R2 receptor subunit. A) GABAB
R2 receptor subunit (aminoacid residues 52-466, record 4F11 in
the PDB database); B) intracellular coiled-coil heterodimer of the
GABAB receptor (record 4PAS in the PDB database).
Р и с. 1. Структура R2-субодиниці ГАМКВ-рецептора.
A B
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5432
A. Yu. NYPORKO, A. M. NAUMENKO, A. GOLIUS, et al.
subunit was divided into three parts, the extracellular
domain, transmembrane one, and intracellular domain,
loaded separately in the Robetta web server. The latter
individually estimated the presence of homologous
fragments and selected an optimal algorithm for
prediction of the protein structure from the ROSETTA
software package implemented in the cluster (in
the absence of homologs, the protein structure is
predicted by the “threading” method). The results
obtained from this server were carefully analyzed in
terms of the consistency with the available data on
the structure and functioning of the GABAB receptor.
First of all, we rejected the models that did not
contain a compact spatial convolution per se, and we
also admitted false those models that contained any
signs of incorrect server operating with the sequence
sent (for example, building the model of a point-
symmetric monomer, lack of the modular protein
organization, packing of the latter in a globule that
hardly contains regular secondary structural elements,
and the presence of a disordered C-terminal “tail”
having more than two hundred amino acid residues).
After preliminary sorting, the models were analyzed
more carefully with respect to clear differences of
domains. The extracellular one had a characteristic
“claw” structure and seven transmembrane helices
forming a transmembrane hydrophobic substitution in
the receptor subunit. The possibility of formation of
a coiled-coil structure in the C-terminal domain was
tested. The structural variants with N- and C-termini
of the protein housed close to each other (according
to the known structural organization of the GABAB
receptor subunits) and those containing more or fewer
transmembrane helices were considered incorrect.
Extracellular Domains of the GABAB Receptor.
Due to the fact that the GABAB R1 and R2 subunits
have a modular architecture, it was possible to predict
the structure of each domain separately and to optimize
the domain geometry irrespectively of the others. The
server operation resulted in making five variants of the
spatial convolution of the extracellular domains of the
R1 and R2 subunits with a given amino acid sequence
that differed in positions of the C- and N-termini
and in convolution of the N-terminus. Thereafter, to
determine the most energetically favorable structure
of the extracellular R1 and R2 subunits for further
modeling, the geometry optimization of those models
was performed, and option A was found to be the most
energetically favorable (Fig. 2.).
Transmembrane Domains of the GABAB
Receptor. Five variants offered by the server differed
from each other in the number of alpha helices.
Similarly to what was performed in modeling of
the extracellular domain of the GABAB R1 and R2
subunits, we geometrically optimized the obtained
models of the transmembrane domain of the R1 and
R2 subunits trying to choose among them the most
energetically favorable one. Option A turned out
F i g. 2. Extracellular domains of
the GABAB R1 receptor subunit
(calculated using the Robetta
server). E are energies of the models
after minimization (optimization
of geometry); for panels A to D,
E= –10248.727123, –9089.999205,
–9096.350844, and –9022.325294
kcal/mol, respectively.
Р и с. 2. Зовнішньоклітинні до-
мени R1-субодиниці ГАМКВ-
рецептора.
A B C
D E
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 433
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
A A
A
B B
B
C
C
D
D
E
E
F i g. 3. Transmembrane domains of the GABAB R1 receptor
subunit, calculated using the Robetta server. E is a local minimum
of the model energy; for panels A to E, Е = –4515.786627,
–4515.786627, –3743.081283, –3865.567809, and –3671.730337
kcal/mol, respectively.
Р и с. 3. Трансмембранні домени R1-субодиниці ГАМКВ-
рецептора.
to be the most energetically favorable (Fig. 3). In
addition to model A, model F also corresponded to
a possible spatial convolution of the transmembrane
receptor (based on the structural criteria), and it had a
less compact arrangement of the helices. However, it
turned out after geometry optimization that the energy
of model F was the greatest among the models studied.
Therefore, the option was rejected.
Intracellular Domains of the GABAB Receptor.
We have also obtained five variants of spatial
convolution of the intracellular domain of the
GABAB R1 and R2 subunits having a given amino
acid sequence. The models differed from each other
in their spatial structure. Models A, D, and F were
inspected to be involved in the formation of the coiled-
coil structure. To determine the most energetically
favorable variant of the model, geometry optimization
was carried out. As a result, option D was defined as
the most energetically favorable (Fig. 4).
Integration of the Modules into the Overall 3D
Structure. The latter was processed using the home-
made FlexBones software. In the models used, GABAB
receptor subunits were dissimilar in the layout of their
extracellular domain. Geometry optimization of the
obtained models was performed to select the most
energetically favorable one for further dimerization
(Fig. 5). The model consists of three domains. In
F i g. 4. Options for the C-terminal domain of the GABAB R1
receptor subunit predicted by Robetta server. E are energies of
the models after minimization (optimization of geometry) for
panels A to E, E = –1711.511183, –1717.739975, –1837.595221,
–2139.205012, and –1789.278895 kcal/mol, respectively.
Р и с. 4. Опції для С-термінального домена R1-субодиниці
ГАМКВ-рецептора.
F i g. 5. GABAB R1 receptor subunits integrated into the overall 3D
structure. E is energy of models after minimization (optimization
of geometry) for panels A and B, Е = –17385.679688 and
–12768.388016 kcal/mol, respectively.
Р и с. 5. R1-субодиниці ГАМКВ-рецептора, інтегровані в про-
сторову структуру.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5434
A. Yu. NYPORKO, A. M. NAUMENKO, A. GOLIUS, et al.
particular, the extracellular domain contains α-helices
and β-elements. The latter sequentially alternate
with α-helices (known as a convolution termed by
Rossmann), thus forming a kind of “claws” linked to a
β-barrel structure. Based on the published data, we can
assume that the neurotransmitter enters precisely the
“claws” site [21]. The transmembrane domain (amino
acid residues 593-854) consists of seven sequentially
connected α-helices. External interfaces of these
α-helices are mainly composed of glycine, leucine,
isoleucine, tryptophan, alanine, serine, and valine
residues, while internal interfaces are composed of
leucine, valine, alanine, serine, cysteine, isoleucine,
tyrosine, asparagine, and glycine. The intracellular
C-terminal domain contains a long helix. The spiral
area (see Table 2) forming a coiled-coil structure is
responsible for dimerization of the receptor subunits.
As the GABAB receptor exists as a dimer, we
decided, after modeling the subunits of this receptor,
to search possible sites of dimerization and spatial
structures of the dimer obtained. Modeling of the
dimeric GABAB R1/R2 subunits was performed
using a SymmDock web server. The latter proposes
several variants of a dimeric structure of the GABAB
receptor, which reflect specific criteria for estimation
and “sorting” of the variants. For example, we may
re-estimate the role of hydrophobic interactions,
electrostatic interactions, balance of hydrophobic
and electrostatic interactions, etc. Using dimerization
servers, we have estimated the type of dimeric
organization of any proposed variant. In particular,
the variants that are basically similar to each other
were ranged, to optimize the number of oncoming
calculations and to choose the one being the most
representative among possible dimeric interfaces.
For the model of the GABAB R1 and R2 subunits
selected, dimeric models have been constructed
(Fig. 6).
From a large number of the structures analyzed,
only one structure corresponding well to the data on
the spatial dimeric organization of GABAB receptor
subunits has been selected, and the contact surfaces
between the subunits in the dimer were studied
(Table 1). Afterwards, the selected dimeric model
was embedded into the biological membrane.
Simulation of the molecular dynamics of the dimeric
complex of the GABAB receptor with the membrane
was analyzed using the Gromass 4.5.3 package. At
that, we observed compaction of the intracellular
domains of the receptor subunits and convergence
of the transmembrane domains on the 18th nsec of
molecular dynamics simulation, as well as interaction
between the extracellular domains of the receptor
subunits on the 36th nsec (Fig. 7). The analysis of
the molecular dynamics over 36 nsec (relative to
the starting geometry) using a g_rms module of the
GROMACS software allowed us to get the values
of RMSD of the dimer geometry of the simulated
complex of the GABAB receptor/bilipidic membrane.
As is shown in Fig. 8.A, the geometry of the model
undergoes major rearrangements within the first
5 nsec of molecular dynamics assessment, and the
value of the latter is 0.2 nm on the 5th nsec compared
to the original model geometry. Then, the changes
occur gradually.
The RMSF of amino acid residues in the dimer were
assessed using the g_rmsf module of the GROMACS
software. It has been shown that the simulated
fluctuations of amino acid residues are virtually
synchronous in both dimeric monomers (Fig. 8.B),
although there are some differences in the RMSF
values for the monomers within the regions having
5-30, 120-145, and 900-925 amino acid residues.
The data obtained suggest that the highest RMSF
values for amino acid residues occur in the N- and
C-terminal regions of the dimeric complex of the
GABAB receptor and the membrane, namely within
regions corresponding to 1-150 and 850-961 amino
acid residues. These regions are the most flexible
and responsible for the N-terminal formation in
extracellular and intracellular domains of the subunit.
In contrast, a region corresponding to 615-850 amino
acid residues is the least flexible, and it is responsible
for the formation of the transmembrane domain.
In addit ion, we have estimated non-valent
interaction energies for the dimeric models using
simulation of the molecular dynamics (36 nsec). As
is shown in Fig. 8.C, the 5-nsec molecular dynamics
simulation and calculated energy were significantly
reduced as compared to the original value, although
these indices somewhat increased afterwards.
Thus, we reconstructed for the first time the full-
size structure of the GABAB receptor and assessed
its behavior under realistic conditions (the GABAB
receptor has been embedded in the bilipidic membrane
with the corresponding salt-water environment). It
was shown that the receptor C-terminal (intracellular)
domains demonstrate a tendency toward compaction,
and coiled-coil areas form a structure almost
identical to that specified by crystallization of these
fragments. It was revealed that extracelullar domains
form asymetric contact interfaces between subunits
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 435
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
T a b l e 1. Amino acid residues of GABAB subunits
Т а б л и ц я 1. Амінокислотні залишки в субодиницях ГАМКВ-рецептора
Amino acid residues of the R1 subunit forming a contact surface Amino acid residues of the R2 subunit forming a contact surface
Intracellular domain
Arg889 Arg787
Glu892 Gln792
Lys893 Asn795
Asn895 His796
Arg896 Arg799
Glu897 Met800
Glu899 Thr803
Lys900 Glu804
Ile901 Asp806
Ile902 Lys807
Ala903 Glu810
Glu904 Glu811
Lys905 Met814
Glu906 Gln815
Glu907 Gln817
Arg908 Asp818
Ser910 Glu821
Glu911 Thr824
Arg913
His914
Gln917
Gln920
Gln921
Arg923
T a b l e 2. Amino acid residues involved in forming of contact interfaces between R1 and R2 subunits after 36 nsec molecular
dynamics
Т а б л и ц я 2. Залишки амінокислот, залучені у формування контактів між субодиницями R1 та R2 через 36 нс
молекулярної динаміки
R1 subunit R2 subunit
Exstracellular domain
Met1 Ser288
Leu2 Gln292
Leu3 Val293
Leu6 His294
Leu7 Thr295
Leu10 Glu296
Phe11 Asn298
Gln21 Ser299
Pro23 Ser300
Asn24 Arg301
Ala25 Cys302
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A. Yu. NYPORKO, A. M. NAUMENKO, A. GOLIUS, et al.
Thr26 Leu303
Ser27 Arg304
Glu28 Lys305
Ile654 Ile490
Met493
Transmembrane domain
Lys590 Leu481
Val597 Ala487
Ser600 Ile490
Leu601 Leu491
Val604 Ile494
Leu605 Ser497
Val607 Ala498
Val608 Phe501
Cys609 Phe502
Ser611 Lys505
Phe612 Asn506
Val854 Arg507
Met857 Asn508
Arg858 Phe762
Leu860 Asn765
Ile861 Gln766
Ser868 Lys510
Lys593
Intracellular domain
Tyr615 Ala783
Asn616 Ser784
Ser617 Ser786
Hsd618 Arg787
Val619 Gln792
Leu860 His796
Arg863 Asp806
Gln867 Lys807
Ala870 Glu810
Gln871 Thr813
Met874 Met814
Asn882 Leu816
Glu885 Gln817
Arg889 Pro820
Glu892 Glu821
Lys893 His830
Arg896 Tyr831
Ala904 Asn839
Hsd914 Glu845
Gln917 Ser846
Ser918 Thr847
Gln921 Asp848
Leu922 Lys851
Glu933
Arg948
Leu949
Asp952
Arg955
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 437
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
Anm
–0.05
–5000
–2000
5000
2000 6000 10000 14000
15000 25000 35000
psec
psec
psec
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0
0
B
C
nm
kg/mol
–320000
–318000
–316000
–314000
–312000
–310000
–308000
–306000
–5000 5000 15000 25000 350000
F i g. 8. Dimeric complex of the GABAB R1 receptor subunit with
the bilipid membrane in the process of calculation of the molecular
dynamics. A) root-mean-square deviations (RMSD) of alpha-carbon
atoms within 36 nsec relative to the starting geometries of the latter
in the modeled dimeric complex of the GABAB R1 receptor subunit
with the bilipid membrane in the process of calculation of the
molecular dynamics; B) root-mean-square deviation fluctuations
(RMSF) of amino acid residues in the simulated dimeric complex
of the GABAB R1 receptor subunit with the bilipid membrane; C)
non-valent interaction of energies in model dimers of the GABAB
R1 receptor subunit with the bilipid membrane at simulation of
the molecular dynamics for 36 nsec. In B, black and gray lines
correspond to the data for chains A and B, respectively.
Р и с. 8. Комплекс R1-субодиниці ГАМКВ-рецептора з біліпідною
мембраною в процесі розрахунку молекулярної динаміки.
F i g. 6. Dimer models for the GABAB R1 receptor subunit
Р и с. 6. Моделі димерів для R1-субодиниці ГАМКВ-рецептора.
A A B
B
C
C
F i g. 7. Changes in the simulated dimer of the GABAB R1 subunit
resulting from the calculation of the molecular dynamics; A, B, and
C) structures before calculation of the molecular dynamics (A),
after 18 nsec (B), and after 36 nsec (C).
Р и с. 7. Зміни в модельованих димерах R1-субодиниці ГАМКВ-
рецептора після розрахунку молекулярної динаміки.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5438
A. Yu. NYPORKO, A. M. NAUMENKO, A. GOLIUS, et al.
R1 and R2 over the 36 nsec molecular dynamics
(Table 2). The structure obtained can be useful for
further examination of the structural mechanisms of
GABAB receptor interaction with GABA agonists and
antagonists, e.g., the contribution of the R2 subunit
to stabilization of the active receptor conformation
and to interaction of the latter with G protein.
It is quite evident that calculation of the molecular
dynamics might be a valuable tool in probing details
of the receptor structure and dynamics. The method of
molecular modeling enables researchers to construct
complete spatial models of any receptor and to meet
challenges in drug discovery and development. For
example, calculations of the molecular dynamics of
ligand-receptor complexes make it possible to predict
and explain the agonist/antagonist location in ligand-
receptor binding sites and to estimate the important
functional significance of amino-terminal domain
dimerization. It also allows one to simulate the
processes of closing and opening of the amino-terminal
domain and propose an alternative explanation for the
functional role of agonists, which consists in changing
the conformations of side chains of the amino acid
residues.
Thus, we succeeded in reconstruction of the 3D
structure of a full-length GABAB receptor in the
real microenvironment. It was shown that a subunit
of the simulated GABAB receptor consists of the
extracellular, transmembrane, and intracellular
domains. The extracellular domain is represented by
β-elements consistently alternating with α-helices
(Rossmann’s convolution) and forming a kind of
“claws” connected by a β-cylinder structure. The
transmembrane domain contains seven concatenated
α-helices, and the intracellular domain consists of
α-helices and a site that serves to form the coiled-coil
structure.
Simulation of the molecular dynamics for 18 nsec
results in compaction of the intracellular domains
of the receptor subunits and in convergence of the
transmembrane domains, whereas simulation of the
molecular dynamics for 36 nsec results in interaction
between the extracellular domains of the receptor
subunits.
Calculations of the RMSF values of simulated
amino acid residues indicate that such fluctuations
are practically synchronous for both monomers of
the dimer. The greatest RMSF values for amino acid
residues are observed in the N- and C-terminal domains
of the simulated dimer complex of the GABAB R1/R2
subunits with the bilipid membrane. The least flexible
site is that located between amino acid residues 615
to 850, and it is responsible for the formation of the
transmembrane domain.
The reconstructed 3D structure of the full-length
GABAB receptor seems to be adequate; in future it will
allow us to simulate the interaction of an agonist with
the examined receptor.
Acknowledgment. The authors are thankful to the Extreme
Science and Engineering Discovery Environment (XSEDE) for
the award allocation number TG-DMR110088.
Since our study dealt exclusively with computer modeling,
it was not necessary to confirm its compliance with the
statements of the International Convention (Strasbourg, 1986,
and later versions).
The authors, A. M. Naumenko, A. Yu. Nyporko, A. Golius,
O. V. Tsymbaliuk, L. M. Shapoval, and T. L. Davidovska,
confirm that they have no conflict of interest with any
organization or person that may be related to this study; there
was also no conflict of interest in interrelations between the
authors.
О. Ю. Нипорко1, А. М. Науменко1, A. Голіус2, O. В. Цимба-
люк,1 Л. М. Шаповал3, T. Л. Давидовська1
РЕКОНСТРУКЦІЯ ПРОСТОРОВОЇ СТРУКТУРИ
ПОВНОРОЗМІРНОГО ГАМКВ- РЕЦЕПТОРА
1 Інститут високих технологій Національного університету
ім. Тараса Шевченка, Київ (Україна).
2 Державний університет Джексона (США).
3 Інститут фізіології ім. О. О. Богомольця НАН України,
Київ (Україна).
Р е з ю м е
Проведена реконструкція просторової структури повнороз-
мірного ГАМКВ-рецептора. Для імітації реального мікро-
середовища ГАМКВ-рецептор був вбудований у біліпідну
мембрану з відповідним водно-сольовим мікрооточенням.
Оскільки гомологічне моделювання ГАМКВ-рецептора є
важливим обчислювальним методом прогнозування про-
сторових координат, коли експериментальні дані щодо
структури не є доступними, ми реконструювали структуру
повнорозмірного ГАМКВ-рецептора з використанням сту-
пінчастого гомологічного моделювання окремих частин
субодиниць. Стабільність субодиниць рецептора оцінюва-
ли, розраховуючи молекулярну динаміку. Було показано,
що субодиниця модельованого ГАМКВ-рецептора склада-
ється з позаклітинного, трансмембранного і внутрішньо-
клітинного доменів. Встановлено, що внутрішньоклітинні
С-термінальні домени рецептора мають тенденцію до ком-
пактизації, а надспіралізовані ділянки утворюють струк-
туру, майже ідентичну до тої, що зумовлена кристалізаці-
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 5 439
THREE-DIMENSIONAL RECONSTRUCTION OF A FULL-SIZE GABAB RECEPTOR
єю цих фрагментів. Проведена реконструкція просторової
структури повнорозмірного рецептора ГАМКВ є адекват-
ною і такою, що може бути корисною для подальшого дослі-
дження структурних механізмів взаємодії даного рецептора
з агоністами і антагоністами ГАМК. Моделювання молеку-
лярної динаміки може бути важливим інструментом вивчен-
ня деталей структури і динаміки рецептора.
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