23730
J. Phys. Chem. B 2005, 109, 23730-23738
Dynamic Mechanism of E2020 Binding to Acetylcholinesterase: A Steered Molecular
Dynamics Simulation
Chunying Niu,† Yechun Xu,† Yong Xu,† Xiaomin Luo,† Wenhu Duan,† Israel Silman,‡
Joel L. Sussman,‡ Weiliang Zhu,† Kaixian Chen,† Jianhua Shen,*,† and Hualiang Jiang*,†,§
Center for Drug DiscoVery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Graduate School,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, Department of Structural
Biology, Weizmann Institute of Science, 76100 RehoVot, Israel, and School of Pharmacy,
East China UniVersity of Science and Technology, Shanghai 200237, China
ReceiVed: September 17, 2005; In Final Form: September 22, 2005
The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving
from the long active site gorge of Torpedo californica acetylcholinesterase (TcAChE) was studied by using
steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding
force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A
that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing
E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with
several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong
direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture
force, ∼550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and
dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating
those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall
are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably,
these aromatic residues, acting in three groups as “sender” and “receiver”, compose a “conveyer belt” for
E2020 entering and leaving the TcAChE gorge.
Introduction
According to the cholinergic hypothesis, Alzheimer’s disease
(AD) is associated with the impairment in cholinergic transmission that leads to memory loss.1 The inhibition of acetylcholinesterase (AChE) activity may be one of the most realistic
approaches to the symptomatic treatment of AD.2 AChE is
responsible for degradation of the neurotransmitter acetylcholine
(ACh) in the synaptic cleft of neuromuscular junctions and of
neuronal contacts in the central nervous system.3 The threedimensional structure of Torpedo californica AChE (TcAChE)4
provided valuable insight for studying the structure-function
relationships of this enzyme. Structurally, there is a narrow
active site gorge about 20 Å deep which consists of two
separated ligand binding sites, the acylation (or active) site and
the peripheral anionic binding site. The acylation site located
at the bottom of the gorge contains residues involved in a
catalytic triad (His440‚‚‚Glu327‚‚‚Ser200) and the important
aromatic residue Trp84. The peripheral anionic binding site is
located close to the mouth of the active site gorge. In this
segment, an aromatic residue, Trp279, is an important element
of this anionic site. At the midway of the deep gorge, a
bottleneck formed by the aromatic side chains of Phe330 and
Tyr121 has a size permitting a water molecule to permeate.
Because the cross section of substrate and inhibitors is much
larger than the size of the bottleneck, large-amplitude fluctua* To whom correspondence should be addressed. Phone: 86-2150806600-1210. Fax: 86-21-50807088. E-mail: hljiang@mail.shcnc.ac.cn.
† Chinese Academy of Sciences.
‡ Weizmann Institute of Science.
§ East China University of Science and Technology.
tions are necessary for substrate or inhibitors to enter or leave.5-7
These structural assignments were confirmed by biochemical
studies, involving site-directed mutagenesis, which attested to
the important role of aromatic residues in AChE proposed on
the basis of the crystallographic data.8,9
Because of the key role that AChE plays in the nervous
system, AChE inhibitors are also used in the treatment of various
disorders such as myasthenia gravis and glaucoma. Their use
has been proposed not only as a possible therapeutic approach
to allay the symptoms of AD but also for the recovery of
neuromuscular block in surgery.4,10 Several clinical studies with
AChE inhibitors such as physostigmine (PHY) and tacrine
(THA)11 have revealed modest improvements in cognitive
function in AD patients. However, some factors still continue
to restrict the acceptance of these drugs, such as variable oral
activity, short duration of action, and side effects.12 E2020
((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) is a piperidine derivative, cholinesterase inhibitor
(ChEI), known by its trivial name donepezil hydrochloride and
marketed as Aricept,13 for the treatment of AD.12 Pharmacological studies have shown that E2020 is a reversible and noncompetitive inhibitor of cholinesterase (ChE) and displays high
selectivity for AChE in comparison to butyrylcholinesterase
(BChE).14,15 It overcomes the deficits of short duration of PHY
and liver toxicity of THA by producing distinct and long-lasting
inhibition of brain AChE and increasing brain content of ACh
in vivo.12,16 The X-ray crystal structure of the E2020-TcAChE
complex revealed a high degree of structural complementarity
between the inhibitor and enzyme. E2020 orientates along the
active site gorge, extending from the active site, at the bottom
10.1021/jp0552877 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/17/2005
E2020 Binding to Acetylcholinesterase
J. Phys. Chem. B, Vol. 109, No. 49, 2005 23731
Figure 1. Ribbon schematic representations of the solvated E2020-TcAChE model for the SMD simulation (water molecules not shown). During
the SMD simulations, E2020 is pulled away from the binding gorge of TcAChE using a harmonic potential symbolized by an artificial spring
connected to the center of mass of the indene ring of E2020. This pulling potential moved with constant velocity Vpull (arrow) along the Z axis. A
structure diagram of E2020 is shown at the bottom right-hand corner. This picture was rendered in the POV-Ray program.18
near Trp84, to the peripheral binding site, at the top near Trp279;
three major functional moieties of E2020, the benzyl group,
the piperidine nitrogen, and the dimethoxyindanone moiety,
make principal interactions with the active site gorge of TcAChE
(Figure 1).17
Different from other AChE inhibitors, such as Huperzine A
(HupA), which binds to the acylation site, E2020 is a typical
bivalent AChE inhibitor that almost orients along the whole
gorge of AChE, capable of binding with both the acylation
(active) site and the peripheral “anionic” site simultaneously.
In our previous study, taking HupA as an example, we
investigated the binding and unbinding processes of acylation
site inhibitors to AChE using the steered molecular dynamics
(SMD) simulation method.19 The detailed analyses for the
structures and interactions in the SMD trajectories detected the
important residues associated with HupA binding and unbinding,
the flip of the peptide bond between Gly117 and Gly118, and
the dynamic effect of the buried water molecules. However,
for bivalent AChE inhibitors, several questions are still open.
How do bivalent inhibitors enter or leave the active site gorge
of AChE? What make bivalent inhibitors cross the narrow
bottleneck of AChE? What roles do aromatic residues play in
the activity and function of the bivalent inhibitors? To answer
these questions, we simulated the unbinding process of E2020
from AChE using the SMD method. SMD is a simulation
method that has been widely used to explore the binding and
unbinding properties of biomolecules and their responses to
external mechanical manipulations at the atomic level.20 In SMD
simulations, time-dependent external forces are applied to the
ligand to facilitate its unbinding from a protein, as shown in
Figure 1. Through the accelerated dissociation process of the
ligand, SMD can reveal information about the protein’s flexibility and its response to the dissociation of the ligand. Analyses
of interactions between the dissociating ligand and the protein
and the relationship between the applied forces and the ligand
position could yield important information about the structurefunction relationships of the protein-ligand complex, the
binding and unbinding pathways, and possible mechanisms of
ligand recognition and inhibition.19,21,22 In the following, we
present the results of SMD simulations for E2020 leaving
TcAChE in water solution. In this simulation study, 26 pulling
velocities were tested to pull E2020 out from the binding gorge
of TcAChE, and corresponding rupture forces and mechanical
works under different pulling velocities were also calculated.
The subsequent detailed analyses based on a SMD simulation
under a velocity of 0.005 Å/ps detected the important residues
associated with E2020 unbinding, revealed the roles that
aromatic residues play in E2020 association and dissociation,
and provided a valid interpretation for pronounced long-lasting
inhibition of this inhibitor.
Models and Methods
The simulation model used in the present study was built up
on the basis of the X-ray crystal structure of the E2020TcAChE complex at 2.5 Å resolution taken from the Protein
Data Bank (PDB),23 entry code 1EVE.17 The missing atoms of
residues Asp2 and His3 were added by using the molecular
modeling software Sybyl 6.7.24 The ionization states of some
residues in TcAChE were determined.25 Residues His440,
Glu443, and Asp392 were neutralized, as pointed out by
McCammon et al.,5 and all other ionizable residues were set as
in their standard protonation states.
All of the simulations were performed with the parallel version of the MD program GROMACS.26 A modification of the
GROMOS-87 force field27-29 was applied for the protein. The
molecular topology file for E2020 was generated by the program
PRODRG30 (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/prodrg.html). The partial atomic charges of E2020 were
determined by using the CHelpG method31 implemented in the
Gaussian 98 program32 at the level of HF/6-31G*. Before the
MD simulations, the E2020-TcAChE complex was solvated
with simple point charge (SPC)33 water molecules in a cuboid
box, in which the shortest distances between the TcAChE surface and the box walls are larger than 10 Å. To ensure overall
neutrality of the system, 43 Na+ and 40 Cl- were added to replace 83 water molecules at physiological concentration in the
box. The simulation system is totally composed of 51 879 atoms.
The LINCE algorithm34 was used to constrain all covalent bonds
to their equilibrium lengths, allowing for a time step of 2 fs.
The system was minimized using the steepest-descents algorithm
to the tolerance of 100 kJ/(mol‚nm). Afterward, the solvent
23732 J. Phys. Chem. B, Vol. 109, No. 49, 2005
Niu et al.
Figure 2. Time dependence of the RMSD from the crystal structure
of the E2020-TcAChE complex for the CR (red) and all atoms (black)
of TcAChE in the 5 ns equilibrium MD simulation.
molecules were heated to 300 K using a 25 ps MD simulation
with E2020 and protein fixed. Following the equilibration, the
protein was relaxed and heated to 300 K using another 25 ps
MD simulation, and finally, E2020 was relaxed to heat up to
300 K by using a 5 ps MD simulation. Finally, the dynamic
simulation of the entire system was carried out for 5 ns under
the normal pressure (1 bar) and room temperature (300 K).
The SMD simulations were performed while the equilibrated
systems were obtained. E2020 was pulled out of the gorge of
TcAChE under different velocities by employing different
artificial harmonic potentials, and the force constant (k) was
set as 280 pN/Å, as shown by the symbolic “spring” in Figure
1. Considering the structural characteristic of E2020, the
harmonic potential was assigned to the center of mass of the
indene ring of E2020, allowing E2020 to walk along the
previously defined axis that extends from atom Ile444-Cδ to
the center of mass of the atomic group of Glu73-CR, Asn280Cβ, Asp285-Cγ, and Leu333-O.19,35
Results and Discussion
Equilibrium Simulation. The structure deviation of AChE
from the initial X-ray crystal structure is assessed on the basis
of root-mean-square deviation (RMSD). It is an important
criterion for the convergence of the protein system. The RMSD
of the CR and all atoms of TcAChE from its crystal structure
versus simulation time are shown in Figure 2. The values of
RMSD are below 0.3 nm, and the structure of TcAChE appears
to have been stabilized after 1.5 ns of equilibration. At the same
time, the energy components and the temperature were inspected
and found to have reasonable stability throughout the 5 ns
simulation (data not shown). Therefore, we may conclude that
the system has been equilibrated after about 1.5 ns, which can
be used as the starting point for the further SMD simulations.
Comparison of Different Pulling Velocities. In general, the
time scale accessible to computer simulation is often much
shorter than the natural time scale of the process in studying
large systems such as biomolecules.36 Therefore, to study the
binding between the protein and ligand, this process should be
accelerated and the pulling velocity (Vpull) should be an
important parameter in the SMD simulation.19 The magnitude
of the pulling velocity will influence the force profile that could
be related to essential rupture events such as the breaking of
hydrogen bonds or hydrophobic interaction between E2020 and
the active gorge of TcAChE. Higher pulling velocity in SMD
Figure 3. (A) Computed unbinding forces as a function of pulling
velocity (Vpull). The inset shows the same data on a logarithmic scale.
(B) Computed mechanical works as a function of pulling velocity (Vpull).
The inset shows the same data on a logarithmic scale. The red points
are the values with the pulling velocity 0.005 Å/ps.
simulation may lead to remarkable nonequilibrium effects, which
may introduce obvious errors into the simulation results.37 The
low-velocity SMD simulation that was carried out on a
millisecond time scale can overcome these disadvantages and
reproduce actual atomic force microscopy experiments; however,
the corresponding computational cost will be very expensive.
To obtain information about the dependency of the rupture
forces on the pulling velocity and to find an appropriate
simulation velocity, 26 different velocities from 0.001 to 0.5
Å/ps were performed in our SMD simulations. E2020, under
the largest pulling velocity of 0.5 Å/ps, only needed 50 ps to
leave the TcAChE gorge completely. However, it took about
20 ns to pull E2020 out of the gorge under a pulling velocity
of 0.001 Å/ps. Figure 3A shows the results of the largest rupture
forces as a function of pulling velocity. The computed rupture
forces as a function of pulling velocity change from 400 to 1100
pN, showing obvious fluctuation, but in general, the maximum
rupture force trends to decrease as the pulling velocity becomes
slower.
To check the influence of friction, the mechanical work
(W(z,V)) done by pulling E2020 out of the gorge from 0 to z
(20 Å, the length of the active gorge of AChE) under the action
of a force, F(z′,V), was calculated by eq 1
W(z,V) )
∫0zF(z′,V) dz′
(1)
E2020 Binding to Acetylcholinesterase
Figure 4. Rupture force (A), numbers for direct hydrogen bonds (B),
atom pairs of hydrophobic interactions (C), and water bridges (D)
between E2020 and TcAChE versus time in the process of E2020
leaving the TcAChE binding gorge. The data in parts A and C are
smoothed over 10 ps intervals.
where F(z′,V) ) k(Vt - z′) is the external force due to the
harmonic constraint at position z′ with pulling velocity V. Figure
3B shows the results of the mechanical works under different
pulling velocities. In general, the work increases as the pulling
velocity increases, because, as expected, more work is dissipated
to conquer larger friction produced by faster pulling velocity
of the moving molecule.
Despite the fact that the pulling velocity 0.001 Å/ps we used
is very slow in the present study (it costs four processors on a
SGI Origin3800 2 months for the simulation), values of the
rupture force and mechanical work of this velocity do not
decrease much in comparison with other velocities. Moreover,
the aim of this study is to address the dynamical processes of
the E2020-TcAChE unbinding rather than reproduce the
accurate binding force or binding free energy. Therefore, in the
following, we use the SMD result under a pulling velocity of
0.005 Å/ps, which produces the lowest rupture force and the
lowest mechanical works with less computer resources, to
discuss the unbinding process of E2020 from TcAChE.
Rupture Force Profile. Figure 4A shows the rupture force
profile in the leaving trajectory of E2020. Different from the
unbinding process of HupA from TcAChE,19 there are four
major peaks in the rupture force profile of E2020; among them,
the highest peak is located at about 2150 ps with the largest
rupture force of ∼550 pN. To elucidate the fluctuation of the
rupture forces along with the E2020 moving trajectory, the
interactions between E2020 and TcAChE in the process of
E2020 leaving the gorge were analyzed. The direct hydrogen
bonds (DHBs), water bridges (WBs), and hydrophobic interactions (HIs) between E2020 and TcAChE were considered. The
interaction features were analyzed by using the tools of
GROMACS26 and the LIGPLOT program.38 The results are
shown in Figure 4B-D. To illustrate the most important residues
of TcAChE concerning E2020 unbinding, six typical snapshot
structures of the E2020-TcAChE complex were isolated from
the SMD trajectory, as shown in Figure 5.
While moving along the binding gorge, interaction between
E2020 and TcAChE changes from one kind to another; for
example, old hydrogen bonds (HBs) break and new HBs form
continuously. Figure 4B illustrates the variation of the direct
HBs (DHBs) formed between E2020 and TcAChE. The number
of DHBs is always less than four. Tyr121, Asn280, Phe284,
Ser286, Phe288, and Arg289 are the main residues forming
J. Phys. Chem. B, Vol. 109, No. 49, 2005 23733
DHBs during E2020 unbinding. From 0 to 300 ps, E2020 forms
DHBs mainly with Tyr121, Phe288, Ser286, and Arg289; from
300 to 1000 ps, only Arg289 forms DHBs with E2020; when
the dimethoxyindanone moiety of E2020 begins to move out
from the peripheral anionic site at about 1000 ps, E2020 forms
new DHBs with Ser286, Asn 280, and Phe284, and this process
holds up to about 1100 ps; then, a DHB vacuum appears from
2160 to 2650 ps; at about 2650 ps, a few DHBs are formed
with the nitrogen of Phe284; no DHBs are observed between
E2020 and TcAChE after 2740 ps. All oxygen and nitrogen
atoms of E2020 take part in the formation of hydrogen bonds,
but mostly, E2020 forms hydrogen bonds with residues of
TcAChE through its carbonyl oxygen atom (O24 in Figure 1)
of the dimethoxyindanone group (Figure 5).
Figure 4C presents the change for hydrophobic interaction
(HI) pairs between E2020 and TcAChE during E2020 moving
through the gorge. In general, the HI pairs are decreasing along
with E2020 leaving away the active binding site. From 0 to
1000 ps, because E2020 occupies most of the gorge (Figure
5A and B), the hydrophobic interaction is relatively stable, as
indicated by the HI pairs. When the dimethoxyindanone moiety
of E2020 moves out of the gorge, the HI pairs decrease
dramatically. After 3000 ps, the HI pairs gradually disappear.
In the whole process of E2020 leaving, the aromatic residues
contribute about 70% to hydrophobic interactions. Table 1 lists
the main residues in the gorge contributing to the hydrophobic
interaction, which indicates that most of the major residues
contributing to the HI are aromatic residues. This may be
assigned to the fact that the walls of the gorge are lined
predominantly by aromatic residue side chains.4,39 The pairs of
HIs demonstrated that Trp84 and Trp279 are the two most
important residues to the hydrophobic interactions between
E2020 and TcAChE; their functions will be discussed below.
Both X-ray crystal structures of inhibitor-AChE complexes4,40-42 and theoretical analyses43,44 have demonstrated the
importance of water molecules in the ligand-AChE binding;
that is, water molecules bridge ligands with AChE. Therefore,
more attention was paid to the water bridges (WBs) between
E2020 and TcAChE during the SMD simulation. The number
of water bridges between E2020 and the residues of TcAChE
through a water molecule is displayed in Figure 4D. Different
from the WBs formed in the HupA-AChE complex,19 E2020
forms WBs mainly with the peripheral residues of the gorge
using two oxygen atoms and one nitrogen atom. From 1000 to
3000 ps, WBs are formed with and break from Ser286, Asn280,
Phe288, and Phe284 (Figure 5C and E). As mentioned above,
these residues are also involved in the formation of DHBs.
Therefore, we conclude that this alternative formation between
DHBs and WBs discourages E2020 dissociation from the gorge
of TcAChE and, reversely, these DHBs and WBs are possibly
the driving force for E2020 binding to TcAChE.
The force peaks and troughs in the force profile obtained from
the SMD simulation clearly reflect the complexity of the
unbinding process of E2020 out of TcAChE, as shown in Figure
4A. There are several major peak regions that correlate with
the break of the DHBs, hydrophobic interactions, and WBs. At
∼270 ps, E2020 forms strongly hydrogen bonds with Phe288
and Ser286; at the same time, the hindrance between the benzene
ring of E2020 and Phe330 prevents E2020 from going through
the bottleneck (a detailed analysis of bottleneck change will be
discussed below) (Figure 5A). All of these produce the first
force peak. However, the rupture force (∼200 pN) corresponding to the first force peak is less than those corresponding to
other high peaks. It is different from the unbinding process of
23734 J. Phys. Chem. B, Vol. 109, No. 49, 2005
Niu et al.
Figure 5. Snapshots isolated from the unbinding SMD trajectory of E2020 at (A) 270 ps, (B) 970 ps, (C) 1615 ps, (D) 2000 ps, (E) 2150 ps, and
(F) 2700 ps. E2020 is drawn in yellow color (oxygen atoms are red, and nitrogen atoms are blue). The dashed lines present hydrogen bonds and
water bridges. Water molecules are shown as a ball model. Hydrogen atoms are not shown.
TABLE 1: Major Residues of TcAChE Forming
Hydrophobic Interactions (HIs) with E2020 and Their Total
Atom Pairs of HIs during the SMD Simulation
atom pairs of
atom pairs of
residues hydrophobic interaction residues hydrophobic interaction
Trp279
Trp84
Leu282
Tyr334
Tyr121
Phe288
Phe330
10477
7911
5663
4910
3657
2912
2676
Phe284
Ser286
Phe331
Phe290
Tyr70
Asn280
Asp72
2627
2228
2191
2157
2096
1928
1622
HupA from AChE, where the highest force appears when HupA
goes through the bottleneck.19 The second force peak appears
at about 970 ps (∼440 pN) when the benzene ring of E2020
passes the bottleneck of the gorge (active site). Breaking of the
hydrogen bond between Arg289 and E2020 and the hydrophobic
interaction between E2020 and TcAChE cause the second force
peak (Figure 5B). At about 2150 ps, most of E2020 is at the
peripheral anionic site of the gorge. At this position, the benzene
ring of E2020 closely contacts with Tyr70, Tyr121, and Trp279.
At the same time, O24 of E2020 forms a DHB and a WB with
Ser286 (Figure 5E). Breaking these interactions produces the
highest force peck (∼550 pN). At about 2700 ps, although most
of the E2020 moiety has moved out of the gorge, the benzene
ring of E2020 still contacts closely with Trp279. Meanwhile, a
DHB is formed between O24 of E2020 and Phe284 (Figure
5F). These interactions discourage E2020 from leaving and
produce the fourth force peak (∼370 pN). It indicates that the
peripheral anionic site of the gorge is the main site that prevents
E2020 from leaving the gorge.
Change of the Bottleneck. Although E2020 orientates along
the active site gorge, the steric hindrance of the bottleneck may
still affect E2020 dissociation. The crystal structures of AChE
revealed that the bottleneck, which mainly is composed of
Tyr121 and Phe330, locates at the middle of the binding gorge.4
The minimal distance between Tyr121 and Phe330 (Y-F
distance) was monitored during the SMD simulation. Figure
6A displays the fluctuations of the Y-F distance during the
SMD simulation. The X-ray crystal structure indicates that the
piperidine ring of E2020 inserts into the bottleneck between
Tyr121 and Phe330,17 forming a cation-π interaction with
Phe330 and a WB interaction with Tyr121. Conformational
E2020 Binding to Acetylcholinesterase
Figure 6. Time dependence of the minimal distance between Tyr121
and Phe330 of TcAChE (A), dihedral angle N14-C17-C18-C23 (B),
dihedral angle C13-N14-C17-C18 (C), dihedral angle C8-C10C11-C16 (D), and dihedral angle C7-C8-C10-C11 (E) of E2020.
The atomic numbers of E2020 are shown in Figure 1.
J. Phys. Chem. B, Vol. 109, No. 49, 2005 23735
change should occur during E2020 leaving away from the gorge
of TcAChE. Accordingly, we monitored the dihedral angles of
the four single bonds connecting the piperidine ring to the
benzene ring (bonds N14-C17 and C17-C18) and dimethoxyindanone (bonds C11-C10 and C10-C8). Figure 6B-E
displays the fluctuations of these dihedral angles during the SMD
simulation.
At the beginning, the benzene ring of E2020 is under the
bottleneck, poised to directly contact with Trp84 at the bottom
of the gorge through π-π stacking. As E2020 moves toward
the outside of the gorge, the benzene ring of E2020 approaches
the benzene ring of Phe330 (from 0 to 350 ps), forming π-π
stacking for a while. Then, the benzene ring of E2020 clashes
with the side chain of Phe330, discouraging E2020 to go through
the bottleneck (Figure 5A). The clash between E2020 and
Phe330 enlarges the entrance of the bottleneck, and the Y-F
distance increases from 0.4 to 0.75 nm (Figure 6A). After the
benzene ring of E2020 crosses the bottleneck, the Y-F distance
recovers to its original value and the bottleneck closes. At ∼3500
ps, although there is no direct interaction between E2020 and
the bottleneck, the bottleneck opens again. This is in agreement
with the conclusion of Tai et al.6,7 and Xu et al.19 that the
bottleneck closing and opening is an intrinsic property of AChE.
In the same time period (0-350 ps), the dihedral angle of bond
C17-C18 (N14-C17-C18-C23) of E2020 flips from -50
to 100° (Figure 6B) and bond C8-C10 (dihedral angle C8C10-C11-C16) rotates from 50 to -50° (Figure 6D); no
dramatical changes occur for the other two dihedral angles. This
Figure 7. Profiles for center-center distances of the benzene ring of E2020 to the aromatic rings of the group 1 residues (Trp84 (d1), Phe330 (d2),
and Phe331 (d3)) (A), group 2 residues (Phe288 (d4), Phe290 (d5), and Tyr334 (d6)) (B), and group 3 residues (Tyr70 (d7), Tyr121 (d8), and
Trp279 (d9)) (C); (D) profiles for the center-center distances between the indene ring of E2020 and aromatic rings of Trp279 (d10), Tyr70 (d11),
Tyr121 (d12), and Phe284 (d13).
23736 J. Phys. Chem. B, Vol. 109, No. 49, 2005
Niu et al.
Figure 8. Major hydrophobic interactions between E2020 and TcAChE in the crystal structure (A) and the snapshots at 250 ps (B), 1350 ps (C),
and 2000 ps (D) during unbinding processes. E2020 is drawn in yellow color (oxygen atoms are red, nitrogen atoms are blue). The dashed lines
are center-center distances between the benzene/indene ring of E2020 and aromatic residues of the gorge of TcAChE.
indicates that both the benzene ring and dimethoxyindanone may
adjust their conformations when E2020 passes the bottleneck,
avoiding serious conflict with Tyr121 and Phe330.
Functions of Aromatic Residues in the Gorge. The above
analyses indicate that aromatic residues are major components
contributing to hydrophobic interactions for E2020 to TcAChE
(Table 1). To further probe the roles that aromatic residues in
the gorge play in the E2020 binding, center-center distances
of the benzene and indene rings of E2020 to the rings of the
aromatic residues listed in Table 1 were monitored during the
SMD simulation; the results are shown in Figure 7. According
to the center-center distances and E2020-AChE interaction
features, aromatic residues in the gorge can be clearly categorized into three groups: group 1 contains Trp84, Phe330, and
Phe331; group 2 consists of Phe288, Phe290, and Tyr334; and
group 3 includes Tyr70, Tyr121, and Trp279. We define the
center-center distances of the benzene ring to the aromatic rings
of these residues as d1-d9 and those of the indene ring to the
aromatic rings of Trp279, Tyr70, Tyr121, and Phe284 as d10d13. By analyzing the profiles of these distances in detail along
the simulation time, an interesting phenomenon has been
addressed that each profile contains platform(s). In general, the
appearing times and durations for the platforms are also
approximately sorted by the groups of the aromatic residues
mentioned above. In the X-ray crystal structure, the benzene
ring of E2020 forms π-π stacking interactions with the aromatic
rings of Trp84 (Figure 8A). After ∼80 ps of simulation, the
aromatic rings of group 1 residues move toward the benzene
ring of E2020; then, the profiles of the center-center distances
d1 and d3 form the first platforms from 80 to 1000 ps and that
of the center-center distance d2 forms a platform from 80 to
1500 ps. During this period, the distances (d1-d3) are relatively
stable at ∼0.5 nm. From 1000 to 1500 ps, the profiles of d1
and d3 form the second platforms, fluctuating slightly around
∼0.8 and ∼0.7 nm, respectively (Figure 7A). Each profile of
the distances formed between the benzene ring of E2020 and
the group 2 residues (d4-d6) also contains platforms. d4 drops
dramatically from 1.3 to 0.8 nm in the period from 0 to 80 ps
and then forms the first platform at ∼0.8 nm until 900 ps, and
d6 forms the first platform at ∼0.7 nm from 80 to 900 ps; then,
these two distances form their second platforms at ∼0.5 nm
from 900 to 2200 ps. d5 decreases from 1.1 to 0.7 nm during
the time period from 0 to 80 ps and then forms a long platform
from 80 to 2200 ps at ∼0.6 nm (Figure 7B). d7 and d9 form
their first platforms from 400 to 1500 ps at ∼1.0 nm and their
second platforms from 1600 to 2600 ps at ∼0.5 nm. The profile
of d8 forms platforms at ∼0.5 nm from 400 to 1000 nm and
from 1600 to 2600 ps, respectively (Figure 7C). Similarly, the
E2020 Binding to Acetylcholinesterase
profiles of center-center distances between the indene ring and
the side chains of Trp279 (d10), Tyr70 (d11), Tyr121 (d12),
and Phe284 (d13) have platforms from 0 to 1000 ps (Figure
7D).
The X-ray crystal structure of the E2020-TcAChE complex
indicates that Trp84 is located near the bottom of the gorge;
Phe330 and Phe331 are positioned near the bottleneck of the
gorge (Figure 8A). After a short-time SMD simulation, the side
chains of these two residues move close to the benzene ring of
E2020. Phe288, Phe290, and Tyr334 are situated around the
middle part of the gorge, and Tyr70, Tyr121, and Trp279 are
laid around the lip of the gorge (Figure 8B). Therefore, the
characters of the center-center distance profiles indicate that
the aromatic residues lining the AChE gorge compose a
“conveyer belt” for E2020 entering and leaving. When E2020
leaves the gorge, the aromatic side chains of the group 1 residues
follow the benzene ring of E2020 moving toward the entrance
of the gorge in the period from 0 to 1500 ps; cooperatively, the
aromatic side chains of the group 3 residues move following
the indene ring of E2020 from 0 to 1000 ps. It seems that these
residues discourage E2020 from leaving. During this period,
the aromatic side chains of the group 2 residues wiggle toward
the benzene ring of E2020 to receive it. From 900 to 2200 ps,
the aromatic side chains of the group 2 residues move along
the benzene ring toward the gorge entrance, and the side chains
of the group 3 residues switch to pick up the benzene ring
(Figure 8C). From 1600 to 2600 ps, the side chains of the group
3 residues send E2020 out following the benzene ring (Figure
8D).
Reversibly, when E2020 binds to AChE, the side chains of
residues consisting of group 3 at the lip of the gorge first
“receive” E2020 through the π-π stacking interaction and then
relay the benzene ring of E2020 to the side chains of the group
2 residues, making E2020 move more deep into the gorge.
Afterward, the side chains of the group 1 residues attract the
benzene ring of E2020 to the bottom of the gorge; at the same
time, the aromatic rings of the group 3 residues fasten the indene
ring of E2020 via π-π stacking interactions; thus, E2020 is
finally fixed inside the gorge.
Conclusions
The present SMD simulations with different velocities have
provided new insight into the unbinding mechanism of E2020
from TcAChE at the atomic level. In the nanosecond simulations, E2020 was pulled out from the long binding gorge of
TcAChE with different velocities, and the unbinding force
profiles were obtained. The simulation results based on the SMD
simulation under a pulling velocity of 0.005 Å/ps have revealed
many dynamic features of the inhibitor unbinding process and
provided insights into the inhibition mechanism of E2020. The
peaks in the force profiles and the SMD trajectory can be
assigned to the forming and breaking of specific direct hydrogen
bonds, hydrophobic contacts, and water bridges. In particular,
our SMD simulations of E2020-TcAChE unbinding allow for
the following conclusions:
(1) Different from other AChE inhibitors such as HupA,19
the major resistance for TcAChE to prevent E2020 from leaving
the gorge is from the peripheral anionic site rather than the
bottleneck. When E2020 arrives at this site, the benzene ring
of E2020 closely contacts with several aromatic residues (e.g.,
Tyr70, Tyr121, and Trp279). Meanwhile, O24 of E2020 forms
a strong DHB and a WB with Ser286 (Figure 5E). These
interactions cause the largest rupture force (∼550 pN) (Figure
4A).
J. Phys. Chem. B, Vol. 109, No. 49, 2005 23737
(2) The rotatable bonds of the piperidine ring to the benzene
ring and dimethoxyindanone are essential for E2020 to go
through the bottleneck of AChE, facilitating E2020 to adjust
its conformation when it passes the bottleneck to avoid serious
conflict with Tyr121 and Phe330 (Figure 6).
(3) As in the binding and unbinding of other inhibitors to
AChE, aromatic residues lining the wall of the gorge play
important roles for E2020 binding to TcAChE. However, the
roles that these aromatic residues play for E2020 binding are
very particular. These aromatic residues are major components
contributing to hydrophobic interactions for E2020 to TcAChE
(Table 1). Remarkably, the aromatic residues lining the TcAChE
gorge compose a conveyer belt for E2020 entering and leaving;
three groups of aromatic residues act as “sender” and “receiver”
successively while E2020 is entering or leaving the TcAChE
gorge (Figures 7 and 8)
Acknowledgment. This work was supported by the State
Key Program of Basic Research of China (grants 2004CB518901
and 2004CB518905), 863 Hi-Tech Program (grants 2002AA233061, 2002AA104270, 2002AA233011, and 2003AA235030),
and Shanghai Science and Technology Commission (grant
03DZ19228).
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