Theory

A Gating Mechanism of the Serotonin 5-HT3

Receptor
Graphical Abstract Authors
Shuguang Yuan, Slawomir Filipek,
Horst Vogel

Correspondence
shuguang.yuan@gmail.com (S.Y.),
horst.vogel@epfl.ch (H.V.)

In Brief
Yuan et al. employ all-atom long-
timescale molecular dynamics
simulations to reveal activation
mechanism of the serotonin 5-HT3
receptor. Serotonin binding first induces
distinct changes in the highly conserved
ligand-binding cage, followed by tilting-
twisting movements of the extracellular
domain, which leads to opening of the
transmembrane helices and the
hydrophobic gate. Finally, the
intracellular helix bundle opens lateral
ports for ion passage.

Highlights
d Rotamer change of W156 in the highly conserved
aromatic cage

d Tilting-twisting movements captured at the extracellular

domain

d Rotamer change of L260 lead to formation of a continuous

water pathway

d Chlorine ions penetrate into the intracellular vestibule

Yuan et al., 2016, Structure 24, 816–825

May 3, 2016 ª2016 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.str.2016.03.019

A Gating Mechanism of the Serotonin 5-HT3 Receptor
Shuguang Yuan,1,* Slawomir Filipek,2 and Horst Vogel1,*
1Instituteof Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
2Laboratory of Biomodeling, Faculty of Chemistry & Biological and Chemical Research Centre, University of Warsaw, Pasteura 1,
02-093 Warsaw, Poland
*Correspondence: shuguang.yuan@gmail.com (S.Y.), horst.vogel@epfl.ch (H.V.)
http://dx.doi.org/10.1016/j.str.2016.03.019
SUMMARY
Our recently solved high-resolution structure of the
serotonin 5-HT3 receptor (5-HT3R) delivered the first
detailed structural insights for a mammalian pentame-
ric ligand-gated ion channel. Based on this structure,
we here performed a total of 2.8-ms all-atom molecular
dynamics simulations to unravel at atomic detail how
neurotransmitter binding on the extracellular domain
induces sequential conformational transitions in the
receptor, opening an ion channel and translating a
chemical signal into electrical impulses across the
membrane. We found that serotonin binding first
induces distinct conformational fluctuations at the
side chain of W156 in the highly conserved ligand-
binding cage, followed by tilting-twisting movements
of the extracellular domain which couple to the trans-
membrane TM2 helices, opening the hydrophobic
gate at L260 and forming a continuous transmem-
brane water pathway. The structural transitions in
the receptor’s transmembrane part finally couple to
the intracellular MA helix bundle, opening lateral ports
for ion passage.
INTRODUCTION
The complex mental capacity and motor behavior of mammals
rely on the evolution of a distinguished central and peripheral
nervous system in which ligand-gated ion channels mediate
fast intercellular signaling at synapses. Pentameric ligand- (i.e.,
neurotransmitter)-gated ion channels (pLGIC) of the Cys-loop
receptor family are the most prominent representatives, and
are also important targets for treating many neuronal disorders
(Corringer et al., 2012; Hurst et al., 2013; Lemoine et al., 2012;
Smart and Paoletti, 2012; Thompson et al., 2010; Walstab
et al., 2010). These receptors function as allosteric signal trans-
ducers across the plasma membrane: at a synapse, a released
neurotransmitter binds specifically at the extracellular site of
its LGIC receptor, inducing multiple conformational changes
to open an intrinsic ion-selective channel (Auerbach, 2014;
Changeux, 2014; Corringer et al., 2012; daCosta and Baenziger,
2013; Schmauder et al., 2011). However, structural and mech-
anistic details of this central process remain speculative, as
directly determined high-resolution structures of mammalian
Cys-loop receptors are rare due to difficulties in heterologous
816 Structure 24, 816–825, May 3, 2016 ª2016 Elsevier Ltd All rights reserved
Structure
Theory
expression, purification, and crystallization of these proteins in
a functional state (Bill et al., 2011).
Present models of Cys-loop receptor structures are based on
electron microscopy images of Torpedo acetylcholine receptor
(AChR) (Unwin, 2013), crystal structures of the bacterial channel
homologs GLIC and ELIC (Bocquet et al., 2009; Hilf and Dutzler,
2008, 2009), a Caenorhabditis elegans glutamate-gated chloride
channel, GluCl (Althoff et al., 2014), and most recently, the first
high-resolution structure of a mammalian pLGIC, the serotonin
5-HT3 receptor (5-HT3R) (Hassaine et al., 2014). Hence, a pro-
totypical pLGIC is composed of three distinct structural and
functional entities: a large b-sheet structured extracellular (EC)
domain comprising the ligand-binding site(s), a helical trans-
membrane (TM) channel-forming part, and an intracellular (IC)
domain that influences ion conductance and interacts with
cellular scaffolding proteins (Thompson et al., 2010). The intra-
cellular domain is missing in the non-mammalian receptor struc-
tures such as GLIC, ELIC, and GluCl and is only poorly resolved
in the Torpedo AChR structure (Unwin, 2013), but is well resolved
in the 5-HT3R structure (Hassaine et al., 2014).
Although the recent high-resolution 3D structures of bacterial
and eukaryotic pLGICs resulted in a common structure fold of
the Cys-loop receptor family, the central question remains un-
resolved, namely, how the binding of an activating ligand at the
EC receptor domain induces the opening of a gate about 60 Å
away in the TM region, allowing the translocation of ions across
the receptor. As experimental details about the structural transi-
tions during channel activation are lacking, allosteric models
have been derived from crystallographic data and molecular dy-
namics (MD) simulations of the different states of the channel
structures of GLIC, ELIC, and GluCl (Aryal et al., 2015; Calimet
et al., 2013; Nury et al., 2010; Sauguet et al., 2014; Zhu and
Hummer, 2012).
Here we have used a total of 2.8-ms all-atom MD simulations
to elucidate at atomic resolution the structural transitions of the
5-HT3R in a lipid bilayer during activation by binding its natural
neurotransmitter serotonin. Our computer experiments started
with the crystal structure of the 5-HT3R in the closed-channel
form (Hassaine et al., 2014), removing the five stabilizing nano-
bodies, embedding the receptor in a lipid bilayer, then inserting
five serotonin molecules into the binding sites of the homopenta-
meric 5-HT3R before finally observing the structural transitions
of the receptor during the activation process toward the open-
channel state. Cryoelectron tomography has shown that the
crystal structure of the 5-HT3R is preserved in lipid bilayers
(Kudryashev et al., 2016). To distinguish ligand-induced confor-
mational changes from stochastic conformational fluctuations,
we performed in addition MD simulations of the apo-5-HT3R,

Figure 1. W156 Conformational Switches and Interaction Network
between Residues in the 5-HT3R
(A) Serotonin in the ‘‘aromatic cage’’ (dotted circle) at the interface between
two neighboring extracellular domains.
(B) Configuration of serotonin in the aromatic cage of the 5-HT3R after 30 ns of
MD equilibration.
(C) Configuration of serotonin in the aromatic cage of the 5-HT3R after 700 ns
of MD simulation.
i.e., the receptor in the absence of an activating ligand. On this
basis our MD simulations delivered a realistic description of
how the binding of the activating neurotransmitter on the extra-
cellular site of the 5-HT3R induces a sequence of conformational
changes leading to the opening of the transmembrane ion chan-
nel, and finally to structural changes at the intracellular site of the
receptor, and are therefore of direct relevance for the under-
standing of neuronal signal transmission on an atomic scale.
RESULTS
Binding of the Activating Neurotransmitter Serotonin to
the 5-HT3R: The Role of W156 and Conserved Motifs
All MD simulations have been performed on the 5-HT3R inte-
grated into a bilayer of 1-palmitoyl-2-oleoyl-phosphatidylcholine
(POPC), as it was shown elsewhere that the channel proper-
ties of the receptor are properly reconstituted in such mem-
branes (Hassaine et al., 2014). By performing 2 3 700-ns
all-atom MD simulations for both the agonist-free (apo) and
the agonist-bound 5-HT3R in a lipid bilayer, we elucidated at
atomic resolution the structural transitions of the receptor during
ligand-induced activation. A wealth of functional studies on the
5-HT3R (Lummis, 2012), the recent high-resolution crystal struc-
tures of the 5-HT3R (Hassaine et al., 2014), and the serotonin-
binding protein in complex with serotonin (Kesters et al., 2013)
allowed us to establish through dedicated docking a highly
reliable model on the binding of serotonin in its 5-HT3R. We
found the bound neurotransmitter to be located in the highly
conserved ‘‘aromatic ligand-binding cage’’ of pLGICs, formed
by Y126, Y207, F199, W156, and W63, at the interface between
adjacent receptor subunits (Hassaine et al., 2014) (Figures 1
and 2), which was very similar to the structure found in the sero-
tonin-binding protein (Figure S1). There is no consensus on how
many serotonin molecules are required to activate the 5-HT3R;
between two and five serotonin molecules have been reported
(Rayes et al., 2009). As the pentameric serotonin-binding protein
(5HTBP) in the crystal structure comprised serotonin in each of
the five binding sites (PDB: 2YMD, 2YME) (Kesters et al., 2013),
we also included five serotonin molecules in the 5-HT3R struc-
ture for simulations. The insertion of serotonin into the ligand-
free 5-HT3R structure (Hassaine et al., 2014) created conforma-
tional stress in the receptor’s binding cage, which initiated
during the first 30-ns equilibration phase structural rearrange-
ments in each of the five ligand-binding cages (Figure 1A): The
serotonin molecule was stabilized via p-p stacking with the
aromatic side chains of Y126 and W156; a cation-p stacking
was established between R65 and serotonin; the aromatic
side chain of W63 was oriented perpendicularly to the aromatic
plane of serotonin, forming a s-p interaction; and a salt bridge
was formed between serotonin’s amine group and E209.
During the subsequent MD simulation period, serotonin and
residues in the aromatic cage underwent further conformational
transitions. While the aromatic moiety of serotonin was partially
p-p stacking between F199 and Y207, the side chain of W156
switched toward a stable position (Figures 1A–1C). Such confor-
mational switches, which are represented in the time course of
c1/c2 angles of W156, took place consecutively in all subunits
of the serotonin-bound receptor (Figure S2). Notably, for the
apo-5-HT3R both c1 and c2 remained unchanged during the
entire MD simulation period (Figure S2).
The 5-HT3R is a prototypical representative of an allosterically
regulated pLGIC where binding of the neurotransmitter serotonin
activates an ion channel via a network of internal conformational
changes (Schmauder et al., 2011; Lummis, 2012). To characterize
the dynamic coupling of the serotonin-binding pocket to other re-
gions of the receptor, we calculated the atomic displacement cor-
relations for each residue pair in MD simulations for the 5-HT3R
without and with bound serotonin. In the correlation network rep-
resentation (Figure 3), nodes correspond to protein residues con-
nected by edges and weighted by the strength of their respective
correlation values. The relation of the correlation network to the 3D
protein structure is also depicted in Figure 3. This method has been
applied elsewhere to dissect allosteric couplings in various sys-
tems (Scarabelli and Grant, 2014; Sethi et al., 2009). Correlation
matrices (Figure S3) show that all loops of the binding pocket (Fig-
ure 2) strongly couple to the extracellular b strands. As indicated in
Figure 3, important correlated motions occur between distinct
structural regions of the extracellular part of the receptor. Binding
of the neurotransmitter serotonin affects collective motions, espe-
cially in loop B (where W156 is located), loop C, the bottom por-
tions of b7-b9-b10, b1-b2-b6, and b10 -b70 -b90 -b100 . While in the
apo-5-HT3R (Figure 3A), the bottom portions of b1-b2-b6 (yellow)
and b7-b9-b10 (orange) form in both depicted subunits one highly
compiled community, they are spread into several groups in the
agonist-bound receptor (cyan, orange, yellow, pink, and white in
Figure 3B). Ligand binding furthermore increased the size of the
black group consisting of loop B, loop C, and the top portion of
b7-b9-b10. These observations strongly indicate that the binding
of serotonin plays a fundamental role in the space of these regions,
inducing an overall looser coupling in the extracellular vestibule.
Importance of Domain Tilting and Twisting for Receptor
Activation
Our MD simulations revealed that activation of the 5-HT3R to-
ward an open channel proceeds in multiple domain movements
Structure 24, 816–825, May 3, 2016 817

in each subunit (Figure 4). To visualize the overall motion within
the receptor we performed a principal components analysis
(PCA), extracting data for each subunit from 1,000 snapshots
evenly distributed over time in the MD simulations (Figure S3).
The channel-gating process visualized by the PCA is best
described as a superposition of radial tilting and tangential
twisting of domains which results in a blooming-like opening of
the receptor channel (Figure 4; Movies S1 and S2). Several mo-
tifs including loop A, loop B, loop C, b1-b2 loop, Cys loop, M2-M3
loop, and the M2 and MX helices follow these motions (Fig-
ure 2A), which are also reflected by the root-mean-square fluctu-
ations (RMSF) over the backbones of the receptor’s subunits
(Figure S4). Interestingly, the M2-M3 loops show by far the
largest RMSF amplitudes in subunits A and E; the M2 helices
in their center show comparable RMSF amplitudes especially
in subunits B, C, and D. This demonstrates the direct coupling
of the motions of M2-M3 loops with the fluctuations of the M2
helices. The importance of ligand-induced loop motions in the
function of pLGICs has been noted by numerous structural and
functional data, as summarized elsewhere (Changeux, 2014;
Corringer et al., 2012; Lummis, 2012; Thompson et al., 2010).
The MD simulations have resolved in detail the temporal
sequence of distinct local structural transitions in the 5-HT3R
during agonist-induced activation. About 30 ns after agonist
binding (during the equilibration period), the b-structured EC do-
mains have finished a rigid-body reorientation resulting in sub-
unit tilting of Dq = 2 –7 out toward helix M4 (Figures 4 and
S5). The tilt angles are slightly different in each subunit but
remain stable during the entire simulation time period, with fluc-
tuations of about ±1 around the mean. The outward tilting in-
duces a substantial repositioning of the b1-b2 loops (5 Å) at
the interface between the receptor’s EC and transmembrane re-
gions, and of loops C (3 Å), which move upward together with
the other extracellular loops (Figure 4). However, in the MD sim-
ulations of the inactive apo form, the tilting angles only fluctuated
within 1 –2 (Figure S5). The structural transitions in the activated
EC domains are complemented by additional twisting motions
(Figure 4B) with characteristic individual time traces. Subunits
A and B reached a final stable state of a twist-angle change of
818 Structure 24, 816–825, May 3, 2016
Figure 2. The Serotonin Binding Pocket of
the 5-HT3 Receptor
Residues lining the serotonin-binding pocket of the
5-HT3R are depicted, together with a conserved
residues logo based on multiple sequence align-
ment on all cationic Cys-loop receptors, including
four 5-HT3 serotonin receptors, 17 nicotinic acetyl-
choline receptors, and one zinc-activated ion
channel. The presence of conserved aromatic res-
idues including W63, Y126, W156, and Y207 imply
that p-p interactions play a central role for binding of
the neurotransmitter. Furthermore, the conserved
negatively charged residues at position 209 indicate
the importance of electrostatic interactions for the
binding of serotonin in the 5-HT3R and beyond for
ligand binding in the case of the other anionic Cys-
loop receptors. The size of the single letters in
the depicted sequence segments scales with its
conservation at this position. Conserved residues
participating in binding serotonin are highlighted by
dashed rectangles.
D4 = 5 –7 after 350 ns, subunit C after 500 ns, and subunits
D and E after 700 ns (Figure S5). By contrast, twist-angle fluc-
tuations in the apo form are distinctly smaller (1 –3 ).
Residues interaction correlation network analysis (Figure S3)
shows that the motions of extracellular part of the receptor are
highly correlated with the M2-M3 transmembrane movements.
In addition, the motions within a subunit couple to the motions
of adjacent subunits (Figure S3). This indicates that the tilting
and twisting in the extracellular receptor part leads to the gating
of the TM channel. Our results are consistent with previous
studies of pLGICs (Calimet et al., 2013) and GABAA receptor
(Miller and Aricescu, 2014).
Pore Expansion during Receptor Activation
In the crystal structure (Hassaine et al., 2014), a constricted ring
of hydrophobic amino acid residues in the central M2 region of
the 5-HT3R blocks the pathway for translocation of water mole-
cules and hydrated ions across the receptor (Figure 5). To allow
the passage of hydrated cations such as Na+, the hydrophobic
constriction site has to function as a gate, which would be able
to open and thus provide more internal space. We compared
the flexibilities of residues in several conserved motifs with the
changes of the inner pore size of the receptor and found that
they are correlated during our MD simulations (Figure 5A). In
the extracellular vestibule of the receptor, structural changes of
loop A and loop B lead to considerable expansion (Figure 5C).
In the closed state of the 5-HT3R the extracellular vestibule
harbors a 4-Å wide constriction, which, after binding of seroto-
nin, increased by 8 Å in diameter during the MD simulations
(Figures 5B and 5C). These changes expand to the interface con-
necting the receptor’s EC domain via b1-b2 and the Cys loops
with M2-M3 loops of the TM domain (Figures 5A and 5B).
In consequence, the central part of the TM pore increases by
2–3 Å in diameter after binding of serotonin (Figures 5C and S7).
The intracellular region of the 5-HT3R is composed of five MA
helices extending from the M4 transmembrane helices. They
form a closed vestibule in the non-activated receptor (Hassaine
et al., 2014) (Figures 5A, 5B, and S8). The binding of serotonin
induces an outward tilting and twisting of the M4 helices, which

Figure 3. Simultaneous View of Community Residue Interaction
Network and 3D Structure of the Neurotransmitter Binding Pocket
of the 5-HT3R
(A) Apo form without serotonin.
(B) With bound serotonin. Colors of the dots in the community analysis
correspond to colors in the indicated regions of the 3D protein structure.
couple to the MA helices to open 10-Å wide lateral portals in the
intracellular vestibule (Figure 5B, right panel; Figures 5C and S7),
allowing the entrance of Cl ions (Movie S3). The Cl ions are first
captured by the positively charged residues K415, R424, and
R428 on the intracellular surface of the receptor, and afterward
move toward R416 inside the intracellular vestibule (Figures S6
and S7). Interestingly, even after binding Cl ions, the pKa values
of these residues remain in the range of 11–13, implying that they
are still protonated. In addition, movements of the ends of the
MA helices form a hydrophobic tunnel, which becomes partly
accessible by water molecules (Figure 5B).
In detail, the following conformational changes in the M4-MA
helix lead to these overall structural changes in the intracellular re-
gion of the receptor (Figure S8). The initial outward bending of the
M4 helix is induced by the local helix bending around residue D434
which in turn breaks the salt bridge D434-R251 within one subunit
and creates a new one, but now between D434 of one subunit and
R306 of the neighboring subunit. The other characteristic changes
occur in the bending of the MA helix around residues D417 and
E418, leading to a break of the inter-subunit salt bridge D417-
K415 followed by a 20 torsion of the MA helix in this region and
the formation of a new inter-subunit salt bridge D417-R416. These
conformational changes occur within all subunits.
All these structural transitions induce substantial volume
changes within different internal pore regions of the receptor:
the volume of the EC vestibule expanded from 39 3 103 Å3 (initial
10-ns MD simulations) by 18%, to 46 3 103 Å3 (690–700-ns MD
simulations), the volume of the TM pore from 2.7 3 103 Å3 by
26%, to 3.4 3 103 Å3, and the volume of the IC vestibule from
7.8 3 103 Å3 by 32%, to 10.3 3 103 Å3.
Molecular Switching of the Hydrophobic Gates L260 and
V264 Open the Ion Channel
Ion-channel gating typically takes place on a submillisecond
timescale (Thompson et al., 2010). The extremely large simula-
tion system of the 5-HT3R, containing 186,500 atoms, cannot
be accessed by classical MD over milliseconds in a conventional
supercomputer (Dror et al., 2012). Instead, we used an advanced
free-energy sampling method, namely well-tempered metady-
namics, to explore the activation process of the 5-HT3R. Meta-
dynamics relies on a history-dependent potential acting on a
selected number of collective variables (CVs) and can substan-
tially speed up sampling and reconstruct the free-energy surface
associated with the CVs (Barducci et al., 2010). To choose the
correct CVs for efficient sampling of the gating process, we first
compared the crystal structure of 5-HT3R in the closed-channel
state (Hassaine et al., 2014) (PDB: 4PIR) with the bacterial pLGIC
homolog GLIC in the open-channel state (Nury et al., 2011) (PDB:
3P50). Two obvious differences were found in the structure
of these two channel proteins. (1) in the open-channel struc-
ture of GLIC in the M2 helix of each subunit, two hydrophobic
residues I233 and A237 adopt different rotamer structures
compared with the hydrophobic residues L260 and V264 at cor-
responding positions in the 5-HT3R (Figure 6). (2) Such differ-
ences influence water and ion penetration in the channel: in
GLIC the I233 residues form a pentagon with a cross-sectional
area of 100 Å2 (Figure 6A), while in the 5-HT3R the L260 resi-
dues span a cross-sectional area of only 40 Å2. Similarly, the
A237 residues of GLIC span a regular pentagon of 118 Å2, but
the corresponding plane of the V264 residues in the 5-HT3R is
only 57 Å2. Therefore, we used side-chain dihedral angles c1 of
L260 and V264 in subunit D as two CVs for well-tempered meta-
dynamics simulations.
Our simulations revealed two characteristic conformations
for both L260 and V264, a closed state I and an open state II
(Figure 6B). The free energies are 17 kJ/mol for state I and
33 kJ/mol for state II. In the closed state I, a hydrophobic
constriction is formed by adjacent pentameric rings of residues
L260, V264, and I268, respectively (Figures 7A and S8A). In the
open-channel state II (Figures 7B and S8A), a continuous water
channel is formed, crossing the hydrophobic constriction site
(Movie S4). Residues interaction network analysis on M2 channel
gating (residues 250–271) show that in the closed state I, all
five submits interacted with each other almost identically (Fig-
ure S8B, left panel). However, in the active receptor state II,
chains A, B, and C are much closer to each other than to subunits
D and E which, on the other hand, are located closely together
(Figure S8, right panel). Consequently, much larger space is
created for movement of water molecules (Movie S4).
DISCUSSION
Here, we compare our findings on the activation process of the
5-HT3R with published data on pLGICs.
Structure 24, 816–825, May 3, 2016 819

(1) Ligand binding. Although the general architecture of the
ligand-binding region of pLGICs has been established
as a result of numerous functional, structural, and
modeling studies (Auerbach, 2014; Changeux, 2014; Cor-
ringer et al., 2012; Hassaine et al., 2014; Lummis, 2012;
Smart and Paoletti, 2012; Thompson et al., 2010), our
MD simulations on the 5-HT3R delivered the first atomic
detailed description of how an activating neurotransmitter
binds to a synaptic pLGIC based on relevant structural
data (Hassaine et al., 2014; Kesters et al., 2013).
(2) Conformational changes in the EC region coupling to the
TM channel. MD simulations have proposed various
models on allosteric transitions in non-mammalian LGICs
(Changeux, 2014). For the GluCl receptor from C. elegans,
MD simulations indicated that channel activation is
induced by opposite movements of the extracellular
versus the TM domains coupled via b1-b2 and M2-M3
loops (Calimet et al., 2013). Despite the similarities with
the 5-HT3R, the two cases are not directly comparable.
The GluCl channel could only be stabilized in an open
state when ivermectin, a synthetic small-molecule allo-
steric modulator, was bound at the interfaces between
TM helices of adjacent subunits, potentiating neurotrans-
mitter binding (Althoff et al., 2014). We, however, have
elucidated the direct activation of a neuronal pLGIC by
its neurotransmitter, which is the actual relevant process
in fast neuronal signal transmission.
(3) Opening the TM channel. A hallmark of pLGICs is the
hydrophobic constriction zone in the center of the TM
channel which, according to the hydrophobic gating
concept, is dehydrated in the closed-channel state
and becomes hydrated when the pore diameter en-
larges beyond a threshold size, allowing water mole-
cules and finally hydrated ions to pass (Aryal et al.,
2015; Beckstein and Sansom, 2006; Zhu and Hummer,
2012). Until now an atomistic description of the gating
process was missing, which we here delivered for the
5-HT3R. We found that an increase in the pore diameter
in the hydrophobic gate is necessary but not sufficient
for hydration and opening of the gate. Our MD simula-
tions revealed that in addition to increasing the pore
820 Structure 24, 816–825, May 3, 2016
Figure 4. Domain Movements and Hydro-
phobic Pore of 5-HT3R
(A) The normal modes of collective motions were
calculated by principal component analysis. The
sizes of the red arrows scale with the amplitudes of
the particular motions.
(B) Global twisting of the receptor’s extracellular
domain by D4 = 5 –7 , indicated by overlaying the
structure of the 5-HT3R before (bold) and after
(faint) binding of serotonin.
(C) Tilting of the b sandwich by Dq = 5 –7 .
diameter, the hydrophobic amino
acids have to perform a confor-
mational switch to drastically
reduce steric hindrance and thus
open the hydrophobic gate for
water passage. This might represent a general mecha-
nism for activating pLGICs.
(4) Conformational changes in the intracellular receptor
domain. Vertebrate pLGICs possess an intracellular
domain of 70–150 residues important for receptor traf-
ficking, clustering at the synapse, and gating (Bouzat
et al., 1994; Thompson et al., 2010; Zuber and Unwin,
2013). In the 5-HT3R this domain is a major determinant
of channel conductance (Kelley et al., 2003; Peters
et al., 2010). Here the MA helices extend from the M4 he-
lices to the intracellular side, creating a closed vestibule
(Hassaine et al., 2014) Potential lateral portals between
MA helices are blocked by the post-M3 loop leaving
only a 3.3-Å narrow tunnel. Further, the MA helical bundle
tightens into a 17-Å-long, 4.2-Å narrow hydrophobic pore.
Therefore, the crystal structure of the non-activated
5-HT3R does not offer an exit pathway for the ions. Our
MD simulations show that the structural changes in the
TM region during channel opening couple to the intracel-
lular MA helices, and induce an opening of lateral portals
at the intracellular vestibule and the formation of a hydro-
phobic pore at the end of the helical bundle. This is the
first mechanistic description of how ions are released
from neuronal pLGICs into the intracellular space.
In summary, we combined classical all-atom, long-timescale
MD simulations with advanced free-energy sampling methods
to explore the activation process of the 5-HT3R integrated in a
lipid bilayer (Figure 8). Our MD simulation study is comparable
with a stopped-flow experiment: We found that the fast binding
of serotonin to the ‘‘aromatic ligand-binding cage’’ exerts a
structural stress to the receptor, which relaxes by multiple struc-
tural transitions toward its activated state. First, the ligand-bind-
ing cage undergoes structural relaxation processes to form
optimal interactions with and integration of the ligand, which
finally induces distinct conformational transitions of W156
sequentially in all subunits. The structural reorganization of the
ligand-binding cage then triggers overall movements in each of
the extracellular subunits, first a 6 tilting followed by 6 twisting,
which appears as a blooming/opening of the entire EC domain
expanding substantially the EC inner pore volume. The EC

rigid-body motions couple via the b1-b2 and the Cys loops to the
M2-M3 loops of the TM region. In consequence, the receptor’s
TM core first expands by an overall outward movement of the
TM helices. The expansion enables the side chains of V264
and L260 of the hydrophobic gate to change conformation to
an open-channel structure, allowing water molecules to translo-
cate from the extracellular to the intracellular vestibule. Later,
beyond our present simulation time, the receptor might enlarge
the water channel further for the passage of Na+ ions across
the constriction site. The structural changes in the TM region
finally couple to the MA helix, inducing a drastic expansion of
the intracellular vestibule together with opening of side portals
for the exit of the cations leaving the TM ion channel and the
entrance of anions.
As our simulations are based on the first high-resolution struc-
ture of a mammalian pLGIC and describe the activation process
of the whole receptor from the extracellular to the intracellular re-
gion, they are of direct relevance for understanding synaptic
signal transmission.
EXPERIMENTAL PROCEDURES
Missing Loop Filling and Refinements
Since the small M2-M3 loop in each subunit was not resolved in the X-ray
structure of the 5-HT3R (Hassaine et al., 2014), the loop refinement protocol
in Modeller (Eswar et al., 2007) V9.10 was used to complete and refine this
structural region. A total of 5,000 loops were generated, and the conformation
with the lowest Discrete Optimized Protein Energy score was chosen for con-
structing the starting receptor structure.
Protein Structure Preparation
All protein models were prepared in the Schrödinger software suite using the
OPLS_2005 force field. Five nanobodies were removed from the crystal struc-
ture (Hassaine et al., 2014) (PDB: 4PIR). Hydrogen atoms were added to the
repaired crystal structure of the 5-HT3R to reflect the physiological pH (7.0) us-
ing the PROPKA (Sondergaard et al., 2011) tool in Protein Preparation tool in
Maestro to obtain the optimized hydrogen-bond network. For this, the con-
strained energy minimizations were performed on the full-atomic models,
with an atom allowed motion of 0.4 Å excluding hydrogens, which were free
to move.
Ligand Structure Preparation
The structure of the neurotransmitter serotonin was obtained from the
PubChem (Wang et al., 2012) online database. The LigPrep module in the
Schrödinger 2014 software suite was introduced for geometric optimization
Figure 5. Flexibility Pattern of a 5-HT3R Sub-
unit and Cross-Sectional View of the 5-HT3R
Channel before and after Activation by Sero-
tonin
(A) Flexibility of particular regions in a single submit
are indicated by wider radius of tube and a red
color. The most flexible regions are colored red,
medium flexible regions white, and least flexible
regions green.
(B) Cross section of 5-HT3R in the non-activated
state before binding of serotonin (left panel) and after
binding of serotonin reaching an open, water-filled
channel state (right panel). Hydrophobic regions are
colored in orange and hydrophilic regions in cyan.
(C) Pore diameter along z axis of the 5-HT3R for the
non-activated receptor state (two independent
MD simulations, black and cyan) and for the open,
water-filled channel state (two independent MD
simulations, red and blue).
using the OPLS_2005 force field. The ionization state of serotonin was calcu-
lated with the Epik (Greenwood et al., 2010) tool employing Hammett and Taft
methods together with ionization and tautomerization tools.
Protein-Ligand Docking
The docking was performed using Glide (Friesner et al., 2004). Based on the
crystal structure of the 5-HT3R (Hassaine et al., 2014), serotonin was docked
into the aromatic cage between two subunits close to W156 in a pose as found
in the crystal structure of a serotonin-binding protein in complex with serotonin
(5HTBP; PDB: 2YMD) (Kesters et al., 2013). Cubic boxes centered on the
ligand mass center with a radius of 8 Å for all ligands defined the docking bind-
ing regions. Flexible ligand docking was executed for all structures. Twenty
poses per ligand out of 20,000 were included in the post-docking energy mini-
mization. We also placed the initial docking pose at random pose and obtained
a similar binding mode as reported previously (Kesters et al., 2013) (Figure S1).
Since the top three were found to be identical with each other, the best scored
pose for the ligand, which was similar to that of previous work (Kesters et al.,
2013), was chosen as the initial structure for MD simulations.
3D Multiple Sequence Alignment
The 3D multiple sequence alignment was done in Strap (Gille et al., 2014),
a JAVA-based tool. It first predicts the secondary structure of each sequence
and aligns them with the specified PDB file afterward. The conservation of
each motif was then submitted to WebLogo for visualization (Crooks et al.,
2004).
Molecular Dynamics Simulations
We modeled the protein, POPC lipids, water molecules, and ions using the
newest CHARMM 36 force field (Klauda et al., 2010), and the ligand using
the CHARMM CGenFF small-molecule force field (Vanommeslaeghe et al.,
2012). The membrane system was built by the g_membed (Wolf et al., 2010)
tool in Gromacs (Pronk et al., 2013) V4.6.5 with the receptor crystal structure
pre-aligned in the OPM (Orientations of Proteins in Membranes) database (Lo-
mize et al., 2011). Pre-equilibrated 234 POPC lipids coupled with 40,760 TIP3P
water molecules and 0.15 M NaCl in a box 100 Å 3 100 Å 3 180 Å were used
for building the protein/membrane system. This resulted in 186,500 atoms in
the simulating system in all. The ligand geometry was submitted to the
Gaussian 09 program (Frisch et al., 2009) for optimization at Hartree-Fock
6-31G* level when generating force-field parameters. The system was gradu-
ally heated from 0 K to 310 K followed by 1 ns initial equilibration at a constant
volume and temperature set to 310 K. Next, an additional 30-ns constrained
equilibration was performed at a constant pressure and temperature (310 K,
1 bar) while the force constant on each atom was trapped off gradually from
10 kcal/mol to 0 kcal/mol. All covalent bond lengths to hydrogen atoms
were constrained with M-SHAKE. van der Waals and short-range electrostatic
interactions were cut off at 10 Å. Long-range electrostatic interactions
were computed by the Particle Mesh Ewald summation scheme. 2 3 700-ns
MD simulations were produced for both the apo form of 5-HT3R and
Structure 24, 816–825, May 3, 2016 821
Figure 6. The Hydrophobic Channel Gate
(A) Comparison of dimensions of the hydrophobic constriction sites (channel
gates) of the 5-HT3R in the closed-channel state (PDB: 4PIR) with that of GLIC
in the open-channel state (PDB: 3P50). Shown are cross sections of the M2 TM
helix of 5-HT3R (green) and GLIC (gray). Five L260 and five V264 residues
(green) in the 5-HT3R were superimposed with corresponding residues I233
and A237 (purple) in GLIC.
(B) Left: free-energy changes in the conformational space of the dihedral an-
gles c1 of L260 and V264 show two distinct minima of the closed (I) and open
(II) channel state in the 5-HT3R. Right: distinct side-chain conformations of
V264 (top) and L260 (bottom) of the 5-HT3R in the closed-channel state before
binding of serotonin (I, green) and in the activated channel state after binding of
serotonin (II, cyan).
agonist-bound receptor. The MD simulation results were analyzed in Gromacs
(Pronk et al., 2013) and VMD (Humphrey et al., 1996). Bio3D (Grant et al., 2006)
was introduced for principal component analysis, and the pore diameters were
calculated by HOLE (Smart et al., 1996). Potential Cl ions entrance pathways
were predicated by Caver3.0 (Chovancova et al., 2012) command line version.
All MD simulations were conducted in Gromacs V4.6.5.
Helix Bending
The bending of a helix with respect to the channel axis was calculated in
Bendix (Dahl et al., 2012) with default settings. Helices were categorized as
linear, curved, or kinked according to their shapes (Bansal et al., 2000). In
the first step we created helical axes for each helix, based on a previously
developed algorithm. Axes are calculated only for helices of a minimal length
of four residues, and a local helix axis was calculated for a residue, based
on its backbone atomic coordinates and those of the three residues succeed-
ing it in the sequence. Accordingly, axes are not calculated for the three
carboxy-terminal residues of the helix. Using these axes the helix is assigned
to a linear, curved, or kinked geometry, according to bending angle criteria be-
tween successive axes (Dalton et al., 2003; Lee et al., 2007).
Hydrophobicity Surface Calculation
The hydrophobicity surface of 5-HT3R was calculated by UCSF Chimera: to
each amino acid residue a hydrophobicity value is assigned according to the
hydrophobicity scale of Kyte and Doolittle (1982).
Pore Diameter and Volume Calculation
The HOLE (Smart et al., 1996) version 2.0 method was used for pore diameter
calculation. The program requires the coordinates of the ion channel of interest
in Brookhaven PDB format. An initial point p, which lies anywhere within the
822 Structure 24, 816–825, May 3, 2016
Figure 7. The hydrophobic Gate of the 5-HT3 Receptor
(A) The cross section perpendicular to the membrane plane through the A and
D subunits of the pentameric 5-HT3R (left panel) in the crystal structure in the
absence of serotonin reveals a closed hydrophobic gate formed by the L260
and V264 residues (cross sections at L260 and V264 planes, right panel)
preventing the entrance of water molecules. Amino acid residues of the M2
helices along the channel are indicated.
(B) A continuous water channel was formed by opening the gate of 5-HT3R by
movements of side chains of L260 and V264.
central channel, is also needed. In addition, the user specifies a vector v in
the direction of the channel axis (referred to as the channel direction vector).
The program reads atoms from the pdb file and sets up a van der Waals radius
for each (various sets are available). The maximum radius R(p) of a sphere
centered at a point p without overlap with the van der Waals surface of any
atom is calculated by
RðpÞ = minNi =atom
11 ½jxi  pj  vdWi ; (Equation 1)
where xi is the position of atom number i, vdWi its van der Waals radius, and
Natom the total number of atoms. The radius R(p) can be regarded as an objec-
tive function of the point p. By using Monte Carlo simulated annealing, adjust-
ing p, the radius of the sphere can be maximized within the pore of the channel.
In all cases p is kept on a plane normal to the channel direction vector v (Smart
et al., 1996).
The grid filling the pore space generated by HOLE (Smart et al., 1996) was
imported into UCSF Chimera for volume calculation. The ‘‘Measure Volume
and Area’’ tool reports the total surface area and enclosed volume of a surface
model. These tools compute surface area and enclosed volume from surface
triangles rather than analytically (Goddard et al., 2007; Pettersen et al., 2004).
Metadynamics Simulations
Free-energy profiles of the systems were calculated using well-tempered
metadynamics in Gromacs (Pronk et al., 2013) V4.6.5 with Plumed (Bonomi
et al., 2009) V1.3 algorithm. Metadynamics adds a history-dependent potential
V(s,t) to accelerate sampling of the specific CVs s(s1,s2,.,sm) (Barducci et al.,
2008). V(s,t) is usually constructed as the sum of multiple Gaussians centered
along the trajectory of the CVs (Equation 2).

Figure 8. Gating Mechanism of 5-HT3R
Side-view cross section through the 5-HT3R shows two protein subunits with
two bound serotonin molecules near the W156 residues in the aromatic ligand-
binding cages. The global quaternary motions (black arrows) induce (1) the
entrance of water molecules (red) into the central transmembrane channel
across the hydrophobic gate represented by V264 and L260, (2) a deeper
penetration of sodium ions (blue), and (3) the entrance of chloride ions (green)
into the intracellular vestibule via lateral portals.
! 512, 333–337.
X
n X
n Y
m
½si ðtÞ  si ðtj Þ2
Vðs; tÞ = Gðs; tj Þ = wj exp 
j=1 j=1 i=1
2s2
Periodically, during the simulation another Gaussian potential, whose loca-
tion is dictated by the current values of the CVs, is added to V(s, t) (Li et al.,
2013). In our simulations, the dihedral angles of W156, c1 and c2, were as-
signed as the CVs s1 and s2, while the width of Gaussians, s, was set to 5 .
Similarly, in the second metadynamisc simulation, the c1 angle of L260 and
V264 were used as CVs. The time interval, t, was 0.09 ps. Well-tempered
metadynamics involves adjusting the height, wj, in a manner that depended
on V(s, t) where the initial height of Gaussians w was 0.03 kcal/mol, the simu-
lation temperature was 310 K, and the sampling temperature DT was 298 K.
The convergence of our simulations was judged by the free-energy difference
between states X and A at 10-ns intervals. Once the resulting data remained
stable over time, the simulation was considered as converged. Each metady-
namics simulation lasted 120 ns, and the results were analyzed upon
convergence.
Residues Interaction Network Analysis
StructureViz (Morris et al., 2007) and RINalyzer (Doncheva et al., 2012), plugin
modules in Cytoscape (Shannon et al., 2003) were used for residues interac-
tion network (RIN) analysis (Scarabelli and Grant, 2014). These modules allow
the interactive analysis of a RIN in a 2D representation of amino acid residues
as nodes and their non-covalent interactions as edges together with the cor-
responding 3D protein structures in UCSF Chimera (Pettersen et al., 2004).
Correlation Network Analysis
Correlated atomic fluctuations of a particular receptor state were character-
ized as reported elsewhere (Grant et al., 2006; Scarabelli and Grant, 2014;
Skjaerven et al., 2014) using Bio3DT. The network nodes represent residues,
which are connected through edges weighted by their constituent atomic cor-
relation values. Community analysis and node centrality with Bio3D and sub-
optimal path calculation with the WISP software (Van Wart et al., 2014) were
performed on each network to characterize network properties and identify
residues involved in the dynamic coupling of distal sites. The parameters for
the suboptimal path analysis included input source and sink nodes, as well
as the total number of paths to be calculated. The latter parameter was set
to 500 paths, which was found to yield converged results in all cases (Scara-
belli and Grant, 2014).
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures and four movies and can be
found with this article online at http://dx.doi.org/10.1016/j.str.2016.03.019.
AUTHOR CONTRIBUTIONS
S.Y. and H.V. initialized the project. S.Y., S.F., and H.V. designed the experi-
ments. S.Y. performed the MD simulations and analyzed the results. S.Y.,
S.F., and H.V. wrote the manuscript.
ACKNOWLEDGMENTS
The major part of computing was done at the Shanghai Supercomputer Cen-
ter. S.F. was funded by the National Center of Science, Poland (grant 2011/03/
B/NZ1/03204). H.V. was supported by the Swiss National Science Foundation
(grant 31003A-133141), the European Community (Project SynSignal, grant
FP7-KBBE-2013-613879), and internal funds of the EPFL.
Received: October 20, 2015
Revised: January 27, 2016
Accepted: March 6, 2016
Published: April 21, 2016
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