Article No. jmbi.1999.3468 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 296, 713±735
Active-site Gorge and Buried Water Molecules in
Crystal Structures of Acetylcholinesterase from
Torpedo californica
Gertraud Koellner1*, Gitay Kryger1, Charles B. Millard2, Israel Silman2,
Joel L. Sussman1 and Thomas Steiner1*
1
Department of Structural
Biology, Weizmann Institute of
Science, Rehovot 76100, Israel
2
Department of Neurobiology
Weizmann Institute of Science
Rehovot 76100, Israel
Buried water molecules and the water molecules in the active-site gorge
are analyzed for ®ve crystal structures of acetylcholinesterase from TorÊ (native enzyme, and four
pedo californica in the resolution range 2.2-2.5 A
inhibitor complexes). A total of 45 buried hydration sites are identi®ed,
which are populated with between 36 and 41 water molecules. About
half of the buried water is located in a distinct region neighboring the
active-site gorge. Most of the buried water molecules are very well conserved among the ®ve structures, and have low displacement parameters,
B, of magnitudes similar to those of the main-chain atoms of the central
b-sheet structure. The active-site gorge of the native enzyme is ®lled with
over 20 water molecules, which have poor hydrogen-bond coordination
with an average of 2.9 polar contacts per water molecule. Upon ligand
binding, distinct groups of these water molecules are displaced, whereas
the others remain in positions similar to those that they occupy in the
native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude ¯uctuations of the loop structures forming the gorge wall. Such
¯uctuations are required to facilitate traf®c of substrate, products and
water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as ``activated'' as
compared to bulk water. This should allow their easy displacement by
incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more ef®cient space-®lling by
substrates and inhibitors may be a major driving force of ligand binding.
# 2000 Academic Press
*Corresponding authors
Keywords: structural water; drug-enzyme complexes; buried water;
hydrogen bonding; aromatic hydrogen bonding
Introduction
The principal role of acetylcholinesterase (AChE,
acetylcholine hydrolase, EC 3.1.1.7) is termination
of impulse transmission at cholinergic synapses by
rapid hydrolysis of the neurotransmitter acetylPresent addresses: G. Koellner, T. Steiner, Institut fuÈr
Chemie-Kristallographie, Freie UniversitaÈt Berlin,
Takustr. 6, D-14195 Berlin, Germany; C. B. Millard,
Toxinology Division, US Army Medical Research
Institute of Infectious Diseases, Fort Detrick, Frederick,
MD 21702-5011, USA.
E-mail addresses of the corresponding authors:
koellner@chemie. fu-berlin.de
[email protected]
0022-2836/00/020713±23 $35.00/0
choline (ACh). In keeping with this requirement,
AChE possesses a remarkably high speci®c
activity, especially for a serine hydrolase, functioning under bimolecular conditions at a rate
approaching the diffusion-controlled limit (Quinn,
1987). Kinetic studies indicate that the active-site of
AChE consists of two major subsites, corresponding to the catalytic machinery and the cholinebinding pocket (Nachmansohn & Wilson, 1951).
The three-dimensional structure of AChE from Torpedo californica (TcAChE) reveals that the catalytic
triad is located near the bottom of a narrow and
Ê deep pocket, named the active-site gorge
20 A
(Sussman et al., 1991). The wall of this gorge is
lined by the rings of 14 highly conserved aromatic
# 2000 Academic Press
714
Water Molecules in Acetylcholinesterase
Figure 1. Topology diagram of TcAChE. Residues forming the active-site gorge are marked as dots and the residues of the catalytic triad are marked as stars. For the amino acid sequence, see Sussman et al. (1991); for location of
the secondary structure elements in the sequence, see Cygler et al. (1993). The disul®de bond in the loop between
strands b3 and b2 is formed by Cys67 and Cys94. Two further disul®de bonds are not shown (Cys254-Cys265 and
Cys402-Cys521).
residues, which may contribute as much as 68 % of
its surface (Axelsen et al., 1994).
A topology diagram of TcAChE is shown in
Figure 1, with the residues forming the active-site
gorge marked as dots (for full assignment of the
structure elements, see Cygler et al., 1993). The
Figure shows that the gorge is formed mainly by
residues of several long loops, with the exception
of the two short a-helices, ab3,2 and a17,8, and
Ala201 at the end of a5,6. The catalytic triad is
formed by residues Ser200, His440 and Glu327,
and the adjacent oxyanion hole is formed by the
main-chain N-H functions of Gly118, Gly119 and
Ala201. Choline recognition involves the sidechains of Trp84 and Phe330, through cation-p interactions (Sussman et al., 1991; Harel et al., 1993; Ma
& Dougherty, 1997) and of Glu199. A pocket
responsible for acetyl ester speci®city (the acyl
pocket) is formed by residues Gly119, Trp233,
Phe288, Phe290 and Phe331 (Harel et al., 1993,
1996). These sites are all located near the bottom of
the gorge. A peripheral choline-binding site,
formed principally by the side-chains of Tyr70 and
Trp279, is located near the gorge opening (Harel
et al., 1993). The upper and lower parts of the
gorge are separated by a narrower section, formed
mainly by the side-chains of Tyr121 and Phe330.
The AChE molecule has a large permanent dipole
moment, aligned with the active-site gorge, which
has been suggested to facilitate attraction of a positively charged substrate down the gorge to the
active-site by the electrostatic ®eld (Ripoll et al.,
1993; Botti et al., 1999). Because the cross-section of
the substrate ACh is much larger than the narrowest part of the gorge, large-amplitude ¯uctuations
of at least part of the gorge wall are necessary to
allow the substrate to enter. Such ¯uctuations are
actually seen in molecular dynamics simulations of
the tacrine-TcAChE complex (Wlodek et al., 1997).
Despite our detailed knowledge of the geometry
of the active-site pocket of AChE, and of the
modes of binding of a variety of inhibitors studied
by X-ray diffraction (Harel et al., 1993, 1995, 1996;
Bourne et al., 1995, Raves et al., 1997; Kryger et al.,
1999; Millard et al., 1999a,b; Bartolucci et al., 1999),
some key features of its enzymatic activity remain
poorly understood. In particular, it remains unclear
how water molecules within the gorge exchange
with the incoming substrate, and how the reaction
products are released from the gorge so rapidly. In
molecular dynamics simulations, temporary opening of parts of the gorge wall were observed,
which would allow at least water molecules to
pass in and out through several ``back doors'' in a
thin part of the gorge wall (Gilson et al., 1994;
Wlodek et al., 1997). These putative alternative
routes to the active-site involve primarily motions
of residues in the -loop formed between residues
Cys67 and Cys94, but also movements of the sidechains of Trp432 and Tyr442. These theoretical
results cannot, however, explain the fast release of
the cationic product, choline. Furthermore, sitedirected mutagenesis experiments do not offer support for the existence of a back door (Kronman
et al., 1994; Faerman et al., 1996).
715
Water Molecules in Acetylcholinesterase
The presence of buried water molecules in
proteins, and their possible role(s), have attracted
the attention of structural biologists (Baker &
Hubbard, 1984), and have been the subject of several statistical studies (Rashin et al., 1986; Williams
et al., 1994; Hubbard et al., 1994). For a set of
homologous serine proteases, a detailed structural
comparison revealed that the buried water molecules were highly conserved (Sreenivasan &
Axelsen, 1992). A number of research groups have
presented evidence that buried water is involved
in local structure stabilization (e.g. see Alber et al.,
1987; Vrielink et al., 1991; Pedersen et al., 1994;
Williams et al., 1994; Shih et al., 1995; Krem & Di
Cera, 1998). Other functional roles have been
suggested. Water-®lled cavities between structural
domains appear to facilitate interdomain motions,
in particular shear motions (Hubbard & Argos,
1996). Buried water appears to permit the presence
of charged side-chains at relatively high concentrations in the interior of proteins (Derewenda et al.,
1994). In cytochrome f, the heme redox center is
linked to a buried water chain that extends to the
electron acceptor, and has been suggested to act as
a ``proton wire'' (Martinez et al., 1996). Possible
roles of buried water molecules in folding and
unfolding of proteins have been discussed (e.g. see
Sundaralingam & Sekharudu, 1989; Buckle et al.,
1996).
In the present study, we analyze structural
aspects of the buried water molecules in TcAChE,
and of the water within its active-site gorge.
Because the X-ray crystal structures available are
Ê , it
all in the medium-resolution range of 2.2-3.0 A
was initially uncertain whether the quality of the
structure models would permit such an analysis.
Normally, hydrogen bonding in biomolecules is
analyzed only for high-resolution structures (better
Ê ). In the present case, however, we have
than 1.5 A
at our disposal ten structures that have been independently re®ned, in which TcAChE is complexed
with various inhibitors. Our analysis rests mainly
on critical comparison of these structures, accepting as valid only observations that are consistent
within the set of ®ve structures with the highest
resolution (see Table 1). It will become evident that
a set of several highly consistent structures of medium resolution allows analysis of hydration effects
with much greater reliability than a single structure
permits, perhaps even a single structure of higher
resolution.
Results
The two classes of water molecules in TcAChE
that are the subject of this study, buried water molecules and water molecules in the active-site gorge,
will be treated separately in the following.
Buried water molecules
Content of buried water
The number of buried water molecules in the
®ve TcAChE crystal structures analyzed ranges
from 36 to 41, corresponding to one buried water
molecule per 13.1 to 14.9 residues. This is a very
high value. Although several proteins are known
with a similar or even higher relative content of
buried water (i.e. water molecules per residue),
more typical values are only half as high (Williams
et al., 1994).
Degree of conservation
In the ®ve TcAChE structures, 45 buried
hydration sites were identi®ed (Table 2A). In none
of the ®ve structures is this set found to be completely populated. Maximum population is found for
2ACK (41 buried water molecules). At 30 sites, a
water molecule is found in all ®ve crystal structures, and is classi®ed as buried in all cases. At
eight sites, a water molecule is present in all structures, but is not classi®ed as buried in all cases
(shown by square brackets in Table 2A). These
water molecules are situated close to the surface of
the protein, and local variations in protein conformation determine whether the site is de®ned as
buried. The seven remaining sites are not populated in all structures. It will be shown below that
Table 1. Number of experimental buried water molecules in structural models of TcAChE
Inhibitor
Fasciculin II
Decamethonium
Native enzyme
Tacrine
TMTFA
Native enzyme
(ÿ)-Huperzine A
E2020
Edrophonium
VX
PDB
code
Resol.
Ê)
(A
Total
water
Buried
water
1FSS
1ACL
1ACE
1ACJ
1AMN
2ACE
1VOT
1EVE
2ACK
1VXR
3.0
2.8
2.8
2.8
2.8
2.5
2.5
2.5
2.4
2.2
36
66
71
82
99
204
252
396
252
339
3
5
15
12
13
36
37
37
41
39
Temp.
(K)
Program
R-value
(%)
Reference
Harel et al. (1995)
Harel et al. (1993)
Sussman et al. (1991)
Harel et al. (1993)
Harel et al. (1996)
273
X-PLOR
19.9
Raves et al. (1997)
273
X-PLOR
20.5
Raves et al. (1997)
100
X-PLOR
18.8
Kryger et al. (1999)
273
REFMAC
21.3
Ravelli et al. (1998)
100
CNS
18.7
Millard et al (1999b)
Ê are all isomorphous
The models are listed in the order of increasing resolution. The ®ve crystal structures with resolutions 42.5 A
Ê,
with 2ACE (space group trigonal P3121, one monomer per asymmetric unit, unit cell constants at room temperature, a ˆ b ˆ 110 A
Ê ; Sussman et al., 1991) and were crystallized under similar conditions at pH 5.8-6.0.
c ˆ 135 A
716
Water Molecules in Acetylcholinesterase
Table 2. Buried and active site water molecules in Torpedo californica acetylcholinesterase
2ACE
1VOT
2ACK
1EVE
1VXR
Cluster
A. Buried water molecules
601
613
602
603
604
607
606
624
607
663
609
605
611
610
612
662
613
636
614
668
617
671
623
664
624
644
626
609
635
635
640
623
641
[633]
647
604
649
655
660
767
[666]
670
718
[675]
711
676
[797]
680
602
681
617
684
601
686
728
687
675
688
734
689
614
694
665
698
689
704
686
709
746
[710]
683
734
650
780
702
795
749
773
-
605
602
613
618
622
626
641
614
601
630
638
604
606
635
620
645
646
609
686
[716]
[647]
648
660
769
610
603
615
639
676
693
681
649
634
658
685
689
672
709
765
756
688
711
807
-
1054
1253
1050
1046
1048
1309
1045
1040
1307
1053
1299
1271
1359
1052
1305
1310
1096
1043
1308
[1185]
[1298]
[1336]
1055
[1180]
1162
1165
1047
1041
1044
1049
1353
1166
1051
1042
1212
1306
1167
[1233]
[1205]
1358
1213
1243
1375
-
1042
1026
1034
1035
1177
1077
1043
1112
1039
1066
1189
1180
1176
1050
1317
1031
[1065]
1028
1059
[1089]
1027
1232
1121
1038
1010
1018
1119
1174
1044
1229
1190
1116
1277
1235
1114
1052
1091
[1282]
[1041]
1278
1194
1254
1337
I (n ˆ 3)
Isolated
V (n ˆ 5)
V (n ˆ 5)
V (n ˆ 5)
IV (n ˆ 4)
II (n ˆ 3)
III (n ˆ 3)
Isolated
I (n ˆ 3)
Isolated
Isolated
Isolated
Isolated
Isolated
IV (n ˆ 4)
Isolated
II (n ˆ 3)
IV (n ˆ 4)
Isolated
Isolated
Isolated
Isolated
Isolated
Isolated
Isolated
V (n ˆ 5)
III (n ˆ 3)
II (n ˆ 3)
V (n ˆ 5)
IV (n ˆ 4)
Isolated
Isolated
III (n ˆ 3)
VI (n ˆ 2)
Isolated
VII (n ˆ 2)
Isolated
Isolated
VII (n ˆ 2)
Isolated
VI (n ˆ 2)
Isolated
Isolated
I (n ˆ 3)
B. Water molecules in the active-site gorge
603
C (Inhib.)
608
O1 (Inhib.)
615
619
618
680
625
611
628
649
633
C (Inhib.)
642
616
678
C (Inhib.)
679
C (Inhib.)
682
612
696
615
719
725
629
727
C (Inhib.)
742
C (Inhib.)
749
677
755
750
767
667
798
707
682
725
733
-
611
617
608
656
743
675
(Inhib.)
643
(Inhib.)
(Inhib.)
627
628
623
621
(Inhib.)
748
701
654
673
752
678
780
-
1158
1163
1164
1235
C (Inhib.)
C (Inhib.)
1159
1254
Ph (Inhib.)
C (Inhib.)
1161
1234
1347
1156
1157
1160
1236
C (Inhib.)
C (Inhib.)
1255
1351
C (Inhib.)
C (Inhib.)
1350
-
1022
1021
1011
1015
1023
1001, 1007
1014
1009
1008
O1 (Inhib.)
1013
1017
1016
1012
C (Inhib.)
1020
1019
1005
1004
1003
1002
1006
1024
C
C
C
C
717
Water Molecules in Acetylcholinesterase
Figure 2. Ribbon representation
of native TcAChE (2ACE) with buried water molecules. Red, conserved buried water molecules;
yellow,
non-conserved
buried
water molecules. Water clusters are
indicated by a translucent envelope
and labeled I to V. The solventaccessible surface of the active-site
gorge is shown in dotted grey.
the degree of conservation is remarkable in regions
close to the active-site gorge, whereas the less conserved buried water molecules are typically found
in regions distal from the gorge.
of the clusters is de®ned in Table 2A. Not all the
clusters are fully populated in all structures, but
the two largest clusters, and two of those with
three molecules, are fully conserved.
Polar contacts suggestive of conventional
hydrogen bonds
Displacement parameters
For the buried water molecules of all ®ve crystal
structures, the polar contacts suggestive of hydroÊ , with A ˆ O, N)
gen bonding (OW A < 3.5 A
were identi®ed. For the example of native TcAChE,
the results are documented in Table 3A. For
hydration sites that are not populated in 2ACE, the
polar contacts in one of the other structures are
given. For the 36 buried water molecules in 2ACE,
the number of polar contacts ranges between two
and ®ve, with an average coordination number of
3.6. In the other four structures, this number is
very similar (Table 4). Of the polar contacts formed
by the buried water molecules, 58 % are with
main-chain atoms of the protein, 24 % with sidechains, and 18 % with other buried water molecules. This parallels the observation that in serine
proteases the buried water molecules form more
polar contacts with main-chain than with sidechain atoms (Sreenivasan & Axelsen, 1992).
Number of isolated water molecules and
water clusters
Of the 45 buried hydration sites in TcAChE, 23
are isolated from the others, i.e. there is no other
water molecule within possible hydrogen bond
Ê ). The other 22 buried sites are found
range (<3.5 A
in clusters. There are seven such internal water
clusters, two consisting of two, three of three, one
of four and one of ®ve water molecules. Labelling
In Table 5, average B-factors are listed for all
protein atoms, for strands b2-b8 of the protein, all
water molecules, and some subsets of water molecules. In all ®ve structures, the 30 conserved buried water molecules have strikingly low average Bvalues, consistently lower than the average B value
of all protein atoms, and only slightly higher than
the average B value of the central b-sheet structure.
Thus these water molecules have very well de®ned
positions, clearly better de®ned than the mobile
parts of the polypeptide chain. The buried water
molecules that are not structurally conserved (site
not buried or not populated in at least one structure) have a higher average B value, but still lower
than the average B value of all water molecules.
(The individual B-values of the buried water molecules are documented in Table S-1 of the Supplementary Material.)
Location in the protein molecule
The locations of the buried water molecules in
TcAChE is shown in an overall view in Figure 2,
using the example of the native enzyme (fully conserved buried molecules shown in red, others in
yellow). Water clusters are shown with a translucent envelope, and the active-site gorge is shown
by its solvent-accessible surface. It is immediately
obvious that the buried water sites are not evenly
distributed over the protein molecule, but that
many are concentrated in a region formed mainly
Table 2. (footnote)
Water molecules in equivalent sites are given on one line. Water molecules in square brackets are present but not buried according
to PRO ACT (Williams et al., 1994).
718
Water Molecules in Acetylcholinesterase
Table 3. Putative hydrogen bonds of water molecules in Torpedo california acetylcholinesterase (2ACE)
Water
A. Buried water molecules
601
Ala157N
602
Ile287O
604
Phe45N
606
Gln68O
607
Glu92O
609
Ser125N
611
Ala152N
612
Gly119O
613
Trp114O
614
Phe155O
617
Leu420N
623
Asp326O
624
Ser226Og
626
Phe155O
635
Asn131O
640
Cys67N
641
Gln500Oe1
647
Tyr70N
649
Tyr96O
660
Arg468NZ1
666
Leu494O
670
Glu37O
675
Ala157O
676
His181O
680
Gly117O
681
Glu199Oe2
684
Gln68N
686
Glu278Oe2
687
Glu278Oe1
688
Pro39O
689
Asn65O
694
Gly441O
698
Phe45O
704
Glu278Oe2
709
Glu5Oe2
710
Ser125O
734
His425O
780
Asp72O
795
Leu177O
(7731VOT)
Tyr442O
Thr193O
(6882ACK)
(7112ACK)
Glu5O
Pro337O
(8072ACK)
Ser228N
(13751EVE)
(13371VXR)
Ser291O
B. Gorge water molecules
603
Ser122OG
608
Gly117O
615
Gly117N
618
Asn85OD1
625
Phe331O
628
Tyr121OH
633
W-742
642
Phe288N
678
W-679
679
W-633
682
Gly118N
696
Asp72OD2
719
Tyr70OH
725
Gln69OE1
727
Trp84O
742
W-633
749
Tyr121OH
755
Arg289O
767
Tyr121OH
798
W-625
Ê)
Distances (A
Contact partners
Gly241O
Ser359O
Arg149NZ1
Val150O
Wat684
Ser125Og
Glu278Oe1
Phe292O
Tyr116N
Val293O
Trp492Ne1
Glu327O
Asn324Od1
Asn167N
Tyr134N
Tyr116OZ
Gln500O
Tyr121O
Ser125Og
Arg468NZ2
Thr496N
Glu37N
His159N
Ile184N
Gly118N
Ser226Og
Wat606
Phe292N
Wat647
Glu92Oe2
Cys94O
Glu445N
Leu156O
Ser291N
Ser28Og
Tyr130O
Ala427N
Phe75N
Leu218O
Wat6501VOT
Thr195N
Ser28Og
Asp351O
Cys231Sg
Wat10421VXR
W-633
Tyr130OH
Tyr130OH
W-749
W-798
W-767
W-603
W-755
W-678
Gly119N
Ser81O
Asp72OD1
Gly123N
W-725
W-618
Ser286O
W-628
W-642
Wat614
Asp399Nd2
Phe153O
Val150N
Wat688
Wat640
Wat687
Wat686
Leu146O
W-601
Pro493O
Ser329Og
Tyr421OZ
Asn167Od1
Leu135N
Gly123O
Arg515N
Glu278Oe1
Wat689
Asn506N
Thr496Og1
Glu49O
Val238O
Gln185N
Phe120N
Gly441N
Wat607
Wat612
Wat611
Wat607
Wat609
Glu445Oe1
Gly166N
Ser291Og
Pro102O
Ser145Og
Wat688
Wat684
Wat689
Lys501O
Phe330N
His440N
Val293O
Phe448O
Wat609
Leu516N
Wat687
Asn506O
Lys501N
Glu49N
Gln185Oe1
Ser122O
Glu443Oe1
Glu508O
Tyr148OZ
Wat704
Wat604
Wat649
W-686
Arg105N
Ser81Og
Glu82Oe2
Thr195Og1
Arg105Ne
Ala477O
Wat6852ACK
Trp279p
Val400O
Wat10661VXR
W-727
Glu199OE1
W-642
W-749
W-679
W-625
W-727
W-719
W-798
Ser200OG
Ala201N
W-628
Ser124OG
W-608
Trp84O
W-603
W-628
W-642
W-727
2.9
2.6
2.7
3.1
2.8
3.0
2.8
2.6
2.8
3.2
3.0
2.7
3.0
2.8
2.5
3.0
3.3
2.9
2.8
3.2
2.6
2.5
2.6
2.7
3.1
2.6
3.1
2.8
3.4
2.8
2.8
2.6
3.1
2.7
2.7
2.7
2.6
3.0
2.7
2.8
2.8
2.7
2.8
3.2
3.1
3.1
2.7
3.2
2.5
3.4
2.6
2.9
2.8
2.7
3.0
3.2
2.7
2.8
2.9
2.6
2.6
2.8
3.3
2.7
2.4
2.7
2.8
2.6
2.8
3.4
2.8
2.7
3.4
2.9
3.4
2.8
2.8
2.8
3.1
3.1
2.9
2.6
2.5
2.9
2.8
3.2
3.1
3.3
3.4
2.5
2.7
3.0
2.5
3.0
2.5
3.0
2.6
3.0
2.6
2.7
2.8
3.1
2.7
2.7
3.1
3.4
2.8
2.7
2.8
3.2
2.8
2.4
2.8
2.8
2.6
2.5
2.5
2.4
3.2
3.0
3.0
2.9
3.4
3.0
2.9
3.1
2.7
2.8
3.3
2.8
3.3
2.6
2.9
3.1
3.2
2.8
2.5
2.5
2.8
2.8
2.9
2.6
3.3
2.6
2.5
2.6
2.5
2.5
3.0
2.7
2.6
3.0
2.9
3.0
2.8
2.9
2.8
2.8
2.5
3.0
3.4
3.2
3.0
3.4
2.6
3.2
2.6
2.7
2.8
2.7
3.1
3.0
2.9
2.7
2.6
2.9
2.5
2.6
3.1
2.7
3.0
2.4
2.7
2.9
2.9
3.3
2.6
2.8
3.3
3.4
2.7
3.3
2.7
3.2
2.5
2.4
3.3
2.8
2.5
2.6
2.8
2.7
2.6
(b)
(a)
(a)
(a)
(a)
(c)
(a)
(d)
(e)
(f)
(a)
(g)
(h)
(i)
(a)
(a)
(j)
(a)
(a)
(k)
(l)
(m)
(n)
(o)
(a)
(p)
(a)
(a)
(q)
(a)
(a)
(a)
(r)
(s)
(t)
(a)
(u)
(v)
(w)
(x)
(y)
(z1)
(z1)
(z2)
(z2)
2.8
3.2
3.2
2.4
3.5
3.2
2.8
2.8
2.9
2.8
3.2
2.8
2.9
Ê . Bold-face, amino acids that are part of the active-site
Putative conventional hydrogen bonds in 2ACE with D(O/N O) < 3.5 A
gorge wall. Water sites given in parentheses are not populated in 2ACE, distances are given for the structure quoted.
(a) Identical hydrogen bond scheme in all ®ve structures. (b) Identical scheme in all structures except 1VXR, where the equivalent
Ê away, and an additional water molecule is inserted with Wat1042 Wat1337 ˆ 2.8 A
Ê . (c) Contact to Ser125of Wat614 is 3.6 A
Ê in 1VOT, 2ACK and 1VXR; additional contact to Asn66Nd2 in 1VOT, 2ACK, 1EVE and 1VXR, with the distance in the
Og > 3.5 A
Water Molecules in Acetylcholinesterase
by loops; the same loops are part of the wall of the
active-site gorge. In particular, all clusters consisting of more than two molecules are located in this
region. Most of these water molecules are perfectly
conserved within the ®ve structures. Many isolated
buried water molecules are adjacent to the bottom
end of the active-site gorge, close to the catalytic
site. Further buried water sites are distributed over
various parts of the enzyme, several poorly conserved ones occurring in distal regions.
Buried water clusters close to the active-site gorge
The most prominent feature in Figure 2 is the
large region of buried water clusters. This water
array is enclosed mainly by long loops (67-94, 117146, 148-167, 279-304, see Figure 1), but also by
two a-helices (a17,8, a37,8), and makes contact with
the central b-sheet structure of the protein at the
beginning of strand b2 and at the end of strand b3.
In this restricted space, there are 22 buried water
sites (i.e. about half of all the buried water molecules), 18 in clusters and four single water molecules. Three of the water clusters (II, III and IV,
with a total of ten water sites) form direct contacts
with amino acid residues forming the active-site
gorge. The other 12 water molecules (clusters I, V,
and the isolated water molecules 626, 670, 675,
698) are not in direct contact with gorge residues.
The polar contacts of all these 22 water molecules
are shown schematically in Figure 3.
Of the clusters contacting active-site gorge residues, the largest is IV, which is formed by a chain
of four water molecules (640 609 689 649).
At one end, this chain is in polar contact
with
the
carbonyl
group
of
Gly123
Ê ). The amino group of
(Wat640 Gly123O ˆ 2.9 A
this residue is part of the active-site gorge, and
forms a possible hydrogen bond with the conserved gorge Wat725 (see below). At its other end,
cluster IV is anchored to the central b-sheet
Ê ; Tyr96 is the amino(Wat649 Tyr96O ˆ 2.8 A
terminal residue of strand b2). Cluster IV also
forms polar contacts with both cysteine residues of
719
the disul®de bridge at the ends of the 67-94 omega
loop, and with several other residues.
Clusters II and III, each consisting of three water
molecules, also contact gorge residues, as shown in
detail in Figure 4. They are arranged around the
charged side-chain of Glu278 in such a way that
four water molecules are in short contact with carboxylate oxygen atoms. Only one position farther
down the main-chain, Trp279 is one of the key aromatic residues near the top of the active-site gorge.
One face of its indole ring forms part of the gorge
wall, and contributes to the peripheral binding site
(Harel et al., 1993). Most remarkably, the other face
of the indole ring of Trp279 is in close contact with
Wat704 of cluster III. The distances of Wat704 to
the six individual C atoms of the benzyl ring are in
Ê , and the distance to the arothe range of 3.4-3.8 A
Ê . These distances, and the
matic centroid is 3.3 A
almost centered face-on geometry, are typical for
aromatic hydrogen bonding of the type XH p(Ph) (Levitt & Perutz, 1988; Desiraju &
Steiner, 1999). At very high resolution, and with
located and re®ned H-atom positions, water-toaromatic hydrogen bonds of similar geometries
have only recently been described in small peptides (Steiner et al., 1998). This particular contact is
well conserved within the ®ve structures studied
(geometries in Table 6); so we feel con®dent that
the interaction Wat704 p(Trp279) actually
represents an aromatic hydrogen bond. Wat612
of cluster III forms a polar contact with another
gorge residue, Gly119O, so that a buried water
bridge is formed between residues of the
peripheral binding site and of the oxyanion hole
(Trp279p Wat704 Wat686 Wat612 Gly119O).
Another buried water bridge between important
gorge residues is formed by cluster II,
Tyr70N Wat647 Tyr121O.
The remaining buried water molecules of the
cluster region have no hydrogen-bonded contacts
with gorge residues (Figure 3(b)), and are not discussed here in more detail. The cluster region as a
whole is remarkably well conserved among the
®ve structures studied. Of the 22 water molecules,
Ê . (d) Identical in all structures except 1VXR, where there are additional contacts to Phe120O (3.3 A
Ê ) and Tyr121N
range 3.0-3.3 A
Ê ). (e) Identical pattern in the other four structures, but the contact to Tyr116N is always shorter in the range 2.9-3.0 A
Ê . (f) Con(3.3 A
Ê in 1VOT; in 1VXR, contact to the equivalent of Wat601 ˆ 3.6 A
Ê , contact to the additional water
tact to Val299O > 3.5 A
Ê . (g) In all other structures additional contact to Ser329N in the range 3.2-3.4 A
Ê ; contact to Ser329Og > 3.5 A
Ê in
Wat1337 ˆ 2.5 A
Ê ), in 1VOT, additional contact to Wat617(6812ACE), 3.2 A
Ê . (i) Contact to
2ACK. (h) In 1VXR, additional contact to Glu443Oe2 (3.1 A
Ê ). (j) Contact to Gln500Oe1 > 3.5 A
Ê in all
Asn167Od1 > 3.5 in all other structures; in 1VXR, additional contact to Wat6142ACE (3.4 A
Ê , and additional contact to the new Wat1316. (k) In all
other structures; in 1VXR the site is not buried, contact to Arg515N > 3.5 A
Ê in 1EVE, and to Arg468NZ2 > 3.5 A
Ê in 1VOT; in
Ê ; contacts to Arg468NZ1 > 3.5 A
structures but 2ACE, contact to Asp504O 3.1-3.4 A
Ê ), and in 1VXR to Wat1311 (3.4 A
Ê ). (l) In 2ACK, 1EVE and 1VXR, additional contact to
1VOT additional contact to Met510N (3.1 A
Ê ). (m) In all other structures, contacts to Glu37N and Glu49O > 3.5 A
Ê . (n) In all other structures, additional contact
Glu500N (3.2-3.4 A
Ê ). In 1VOT, contact to Ile184N > 3.5 A
Ê . (p) In 1VOT, additional contact to Wat6242ACE (3.2 A
Ê ). (q) In 2ACK,
to Arg242N (3.2-3.5 A
Ê and additional contact to Glu68O (2.9-3.2 A
Ê ). (r) In 1VXR, additional contact to
1EVE and 1VXR, contact to Glu278Oe1 > 3.5 A
Ê . (s) In 1VOT, 2ACK and 1EVE, contact to Ser291Og > 3.5 A
Ê . (t) In 2ACK, 1EVE and 1VXR: contact to the additional
Pro165N, 3.4 A
Ê ); in 1VOT and 2ACK, contact to additional water
water 7112ACK. (u) In 1EVE and 1VXR, additional contact to Tyr442O (3.0-3.2 A
Ê ). (v) In 1EVE and 1VXR, additional contact to Asp72Od1 (2.8-3.4 A
Ê ); in 1EVE, 1VOT and 1VXR, contact to
Wat7731VOT (2.7-2.9 A
Ê ; in 1EVE, contact to external water Wat12321EVE (2.9 A
Ê ). In 1EVE and 1VXR, contact to external water molecules
Glu82Oe1 > 3.5 A
Ê ). (y) In 1EVE, additional contact to Val194N (3.1 A
Ê ).
(Wat12041EVE, Wat1193VX). (x) In 2ACK, additional contact to Glu445Oe2 (2.5 A
(z1) Identical scheme in the structures where the site is populated. (z2) Populated in only one structure.
720
Water Molecules in Acetylcholinesterase
Ê for buried and gorge water molecules
Table 4. Average number of polar contacts shorter than 3.5 A
Buried
Gorge
2ACE
1VOT
2ACK
1EVE
1VXR
3.6
2.9
3.6
3.1
3.8
3.3
3.6
3.3
3.8
2.7
19 are fully conserved (i.e. always present and
always buried). One site of cluster I is not always
populated (Wat1137 found only in 1VXR, not
shown in Figure 3(b)). The isolated water
molecules 670 and 675 are always present but not
always considered as buried.
Other buried water molecules close to the
active-site gorge
Apart from the region of buried water clusters,
several other buried water molecules form polar
contacts with residues of the active-site gorge (602,
623, 680, 681, 694, 710, 780, Figure 3(c)). A striking
water molecule is Wat623, which bridges the mainchain groups of Glu327 and His440 of the catalytic
triad Glu327O Wat623 His440N, and forms
another hydrogen bond with an important gorge
residue, Wat623 Phe330N. The peptide N-H of
Glu327 forms a hydrogen bond with a conserved
external water molecule in a surface cleft,
Glu327N Wat673, showing that the gorge wall is
quite thin in this region. Water molecules 681 and
694 interact with gorge residues neighboring the
catalytic triad, Glu199Oe2 Wat681 Gly441N,
and Wat694 Gly441O.
Slightly separate from the catalytic triad, Wat680
is tightly coordinated by main-chain atoms of the
gorge residues Gly117O, Phe120N and Ser122O.
This molecule is located immediately ``behind'' the
wall of the gorge, and is indirectly connected with
the gorge Wat608 through the hydrogen bond
chain Wat680 Gly117O Wat608. Three other
buried water molecules, Wat602, Wat710 and Wat
780, are in polar contact with gorge residues, but
relatively distant from the catalytic site, and are
not described in greater detail.
Of the seven water molecules described here,
®ve are fully conserved in the ®ve structures studied. The exceptions are Wat710 (not classi®ed as
buried in 2ACE), and Wat780 (not classi®ed as buried in 1EVE and 1VXR).
Other buried water molecules
The ®nal class of buried water molecules are
those outside the cluster region, and not close to
the active-site gorge. These water molecules are
loosely scattered over the rest of the protein, and
show no obvious concentrations. Their polar contacts are shown in Figure 3(d). Many of these
water molecules are near the protein surface, and
are poorly conserved.
The water molecules in this group typically have
well-de®ned roles in local structure stabilization.
For example, they stabilize secondary structure
elements and loops. As a single example, the polar
interactions of Wat613 are shown in Figure 5. This
water molecule is located at the ends of the parallel
strands, b3 and b4, between Tyr116 and Leu146.
By forming the hydrogen-bonded bridge
Tyr116N Wat613 Leu146O, the ends of the
strands are still linked. Related functions at the
ends of b-sheet structures are also assumed by the
buried Wat617 (strands b8 and b9), Wat641 (strands
b9 and b10) and Wat709 (strands b1 and b2). Some
buried water molecules are found at the ends of ahelices (Wat635 at a3,4 and Wat678 at a4,5), and
others stabilize otherwise irregular loop regions
(Wat660 in loop 505-510, Wat666 in loop 479-502).
No correlation is apparent with the asymmetric
charge distribution
As already mentioned, it has been suggested
that one role of buried water molecules is to facilitate placing of charged residues in the interiors of
proteins (Derewenda et al., 1994). For AChE, it is of
particular interest to analyze the distribution of
buried water molecules in this context, due to its
unusually high asymmetry in charge distribution
(Ripoll et al., 1993; Felder et al., 1998).
At physiological pH, TcAChE has 48 positively
charged and 59 negatively charged side-chains,
with His residues assumed to be uncharged. Thus
the net charge is ÿ11e, with negative charges concentrated preferentially in the ``Northern hemi-
Ê 2) in the ®ve crystal structures analyzed (average values and in some lines also s)
Table 5. B-Factors (A
Protein, all atoms
Protein, strands b2-b8,
Water molecules, all
Buried water, conserved
Buried water, non-conserved
Gorge water
2ACE
1VOT
2ACK
1EVE
1VXR
24
19
33
20/5
28/4
27/10
24
18
33
22/7
26/6
26/8
37
30
48
31/7
40/6
38/12
28
20
37
22/8
33/10
22/10
38
32
45
32/4
39/7
39/11
721
Water Molecules in Acetylcholinesterase
Table 6. The water-aromatic contact Wat7042ACE Trp279
2ACE
1VOT
2ACK
1EVE
1VXR
Ê)
OW C range (A
Ê)
OW M (A
C-atom closest to Wat
3.4-3.8
3.4-3.7
3.2-3.7
3.2-4.0
3.1-3.7
3.3
3.3
3.2
3.4
3.1
CZ
CZ
Cz3
Cz3
Cz3
The target is the six-membered ring, M ˆ aromatic midpoint.
sphere'' and positive charges in the ``Southern
hemisphere'' (North and South with respect to the
orientation viewed in Figure 2). To see if the occurrence of water clusters is correlated with charge
concentrations, the number of charges per amino
acid residue is listed in Table 7 for several relevant
regions, and for the protein as a whole. It can be
seen that the Northern loops enclosing the water
clusters (67-94, 117-131, 148-167, 227-304) possess a
density of charged side-chains similar to that of the
protein as a whole. In contrast, the Northern region
325-383, which has an even higher charge density
(0.26 charged side-chain per residue), hosts only a
few buried water molecules. Thus, there is no
obvious correlation between the distributions of
charges and of buried water molecules.
pation by species other than water cannot be
excluded (Axelsen et al., 1994; Raves et al., 1997).
Furthermore, the conformations of some important
side-chains contributing to the gorge wall respond
to the shape and nature of the inhibitor, speci®cally, those of Trp279 and Phe330 (Harel et al.,
1993; Kryger et al., 1999). This necessarily affects
the position of neighboring water molecules.
Finally, it appears that for the gorge water molecules, the resolution and re®nement strategy are
more critical than for the buried water molecules.
Thus, the structure of highest resolution, 1VXR
Ê ), shows features that cannot be discerned in
(2.2 A
Ê resolution.
the structures at 2.4-2.5 A
Water molecules in the active-site gorge
Putative conventional hydrogen bonds formed
in the active-site gorge are listed in Table 3B for
native TcAChE, using the cutoff criterion
Ê . (The corresponding numerical data
O A < 3.5 A
for the inhibitor complexes are given in Tables S-2
to S-5 of the Supplementary Material.) The interaction patterns are shown schematically for all ®ve
structures in Figure 7.
In the gorge of native TcAChE there are several
water arrays between which there is no polar conÊ . These arrays are indicated by differtact of < 3.5 A
ent colours in Figure 7. The number of polar
contacts per molecule is clearly lower in the gorge
than for the buried water molecules (Table 4).
Thus, in 2ACE, the gorge water molecules are
involved in 2.9 conventional polar contacts on the
average, as compared to 3.6 for the buried water
molecules. The poor hydrogen bond coordination
presumably indicates an activated state of these
water molecules; thus incoming molecules, e.g. the
substrate, ACh, could easily displace them. Never-
Polar contacts suggestive of hydrogen bonds
In the ®ve crystal structures studied, a total of 28
de®ned hydration sites were located within the
active-site gorge, as shown in Table 2B (at the
upper end of the gorge, a boundary between the
gorge and the outside can be drawn only somewhat arbitrarily). Most of the water molecules are
structurally conserved, unless they are replaced by
a bound inhibitor. The total water occupancy
found in the gorge ranges from 15 water molecules
located in 1VOT to 20 located in 2ACE, and 22 in
1VXR. An overall view of the gorge water
molecules in 2ACE is shown in Figure 6.
The water molecules in the active-site gorge are
more dif®cult to compare among the different
structures than the buried water molecules. One
reason is the fact that different inhibitors displace
different water molecules. Another reason is that
part of the gorge water seems to be disordered.
Moreover, for certain hydration sites, partial occu-
Table 7. Charged side-chains in some segments of Torpedo california acetylcholinesterase
Sequence
Whole protein
67-94 (
-loop)
117-131
148-167
227-304
325-383
424-444
480-501
Positive
Negative
Net charge
All charges
Charged groups
per residue
Contact with
cluster region
48
1
0
1
8
4
1
3
59
5
1
1
12
11
2
3
ÿ11
ÿ4
ÿ1
0
ÿ4
ÿ7
ÿ1
0
107
6
1
2
20
15
3
6
0.20
0.22
0.07
0.11
0.26
0.26
0.15
0.29
Yes
Yes
Yes
Yes
No
No
No
722
Water Molecules in Acetylcholinesterase
Figure 3. (Legend opposite)
theless, the active-site gorge also contains some
tightly coordinated water molecules.
Displacement parameters
The average B value of the gorge water molecules is clearly larger than for conserved buried
water molecules, but is still smaller than the average for all water molecules (Table 5). It is of interest that within the set of gorge water molecules,
the variation in B values is very large. In 2ACE, for
example, the B values of the buried water molÊ 2,
ecules are relatively homogeneous, with s ˆ 5 A
whereas the B values for the gorge water molecules
Ê 2. This indicates
are widely scattered, with s ˆ 10 A
that there is a wide variation in water stability
within the gorge; some water molecules occupy
well coordinated and stable positions, whereas
others appear to be very mobile, and are thus
characterized by high B values.
Water Molecules in Acetylcholinesterase
723
Figure 3. Representations of the secondary structure of TcAChE and of the polar contacts of buried water molecules. (a) The part of the cluster region that is in contact with residues of the active-site gorge; (b) the part of the
cluster region that is not in contact with gorge residues: in this region there are also four isolated water molecules; (c)
the isolated water molecules in polar contact with residues of the active-site gorge; (d) water molecules that are
neither in the cluster region nor have contact with gorge residues.
Water arrangement in the gorge of native TcAChE
Ten water sites can be seen in the lower part of
the gorge of 2ACE (shown in blue in Figures 6 and
7(a)). An isolated water molecule, Wat682, is
tightly coordinated to the residues in the oxyanion
hole, Gly118N, Gly119N, Ala201N, and to Og of
the active-site serine residue, Ser200. This is a
peculiar hydrogen bond coordination, since the
three peptide N-H groups can act only as donors,
so that Ser200Og is the only acceptor nearby for the
two OH groups of the water molecule. The closest
water molecule is Wat742, which is at a distance of
Ê from Wat682. This does not de®nitely
3.8 A
exclude hydrogen bonding, but such a hydrogen
bond can only be a poor one. Another isolated
water, the well conserved Wat615, is located in a
pocket at the bottom of the gorge. In the huper-
724
Water Molecules in Acetylcholinesterase
Figure 4. Ball and stick representation of the portion
of the 2ACE structure containing clusters II and III.
Note
the
putative
aromatic
hydrogen
bond
Wat704 p(Trp279). Cluster II is drawn in pink, and
cluster III in blue.
zine-TcAChE complex, the equivalent of Wat615
makes a polar contact with the bound inhibitor
(see below), clearly showing that it is accessible
from the gorge.
Figure 5. Ball and stick representation of strands b3,
b4 and b6 in 2ACE. The isolated buried Wat613, holding
together the ends of strands b4 and b3, is shown as a
large red ball.
Figure 6. Ribbon diagram of the water-®lled activesite gorge of 2ACE. The solvent-accessible surface is
shown in dotted grey. Color coding of the individual
water molecules, shown as balls, relates to the grouping
shown in Figure 7(a).
The remaining eight water molecules in the bottom part of the gorge form an extended cluster of
irregular shape. Wat725 is placed in a pocket, and
makes ®ve conventional polar contacts, four with
gorge residues and one with a water molecule. The
other seven water molecules form an array that is
connected to the gorge wall only loosely. Only one
of these molecules forms two polar contacts with
the gorge wall (Wat608), two make one contact
(Wat678, Wat727), and four are engaged in polar
contacts only with other water molecules (Wat603,
Wat633, Wat727, Wat742). Three of the water sites
are very poorly coordinated, with two polar contacts found for Wat678 and Wat679, and only one
for Wat742. This suggests disorder and/or uncertainties in the structural model. Notably, the very
poorly coordinated Wat742 is the water molecule
that is closest to the acyl pocket (see below).
In the upper part of the gorge, two distinct
water arrays are found. The larger of the two
(shown in red in Figures 6 and 7(a)) extends from
the narrow middle part towards the gorge
opening. The shortest contacts to the water
molecule in the lower part of the gorge are
Ê , Wat749 Wat633 ˆ 3.9 A
Ê,
Wat618 Wat603 ˆ 3.7 A
Ê
and Wat749 Wat603 ˆ 4.0 A. These are not very
long distances, and in some other structures there
are direct polar contacts between water molecules
in the upper and lower gorge sections. In 2ACE,
725
Water Molecules in Acetylcholinesterase
Wat696 seems to be isolated from all other gorge
Ê
water molecules, with the shortest contact at 3.9 A
to Wat618. However, in all other structures,
Wat696 and Wat618 are in hydrogen bond distance
Ê ), so that we assume that also in
(range 2.7-3.5 A
native TcAChE, Wat618 is not isolated in reality.
Finally, a group of four water molecules is found
close to the opening of the gorge (shown in green
in Figures 6 and 7(a)). Hydrogen bonds are formed
with the carbonyl groups of Phe331, Arg289 and
Ser286, and with the peptide NH of Phe288. The
shortest contacts of this array to the water molÊ,
ecules shown in red are Wat767 Wat642 ˆ 4.0 A
Ê
and Wat767 Wat798 ˆ 4.6 A.
Water contacts of the aromatic gorge residues
in 2ACE
As much as 68 % of the gorge surface is contributed by aromatic side-chains (Axelsen et al., 1994),
which are extensively involved in speci®c interactions with both substrates and inhibitors
(Sussman et al., 1991; Harel et al., 1993, 1996; Raves
et al., 1997; Kryger et al., 1999). It is of interest to
see how these formally hydrophobic groups interact with the solvent molecules in the water-®lled
gorge. Apart from van der Waals interactions, aromatic side-chains can be engaged in two kinds of
weak directional interactions with water molecules.
These are C-H OW interactions involving the
ring edges, and OW-H Ph interactions involving
the ring faces (surveyed by Desiraju & Steiner,
1999). Upon close inspection, however, only a
small number of such interactions seem to occur in
the gorge of 2ACE.
In the choline-binding site, the face of the sixmembered ring of Trp84 has two water molecules
in its wider vicinity, Wat678 M ˆ 4.4 and
Ê (M ˆ midpoint of the sixWat679 M ˆ 4.7 A
membered ring). This arrangement will be shown
below in more detail for 1VXR. Wat679 is also relatively close to the phenyl ring of Phe330,
Ê . The four aromatic groups of
Wat679 M ˆ 4.6 A
the acyl pocket have no short contact to water
molecules. Thus, Trp233, Phe288 and Phe290 have
Ê of the midpoint
no water molecule within 5.0 A
of their six-membered rings, and the closest contact
of
Phe331
with
a
water
molecule
is
Ê . This means that the surface
Wat742 M ˆ 4.7 A
of the acyl pocket is actually avoided by water
molecules, at least by those that can be assigned in
the experimental X-ray structure. The side-chain of
Trp279 faces a part of the gorge that is ``empty'' in
2ACE, but water molecules have been located
there in 1VOT, 2ACK and 1VXR. For the example
of 1VXR, it will be shown below that gorge water
molecules are actually close to the face of Trp279.
In 2ACE, the side-chain of Tyr70 probably accepts
an aromatic hydrogen bond from Trp279Ne-H,
which is well conserved in the other structures
(Table 8). The aromatic rings of Tyr121 and Tyr130
have no short contact with water molecules. No
example of clear and conserved C-H OW hydrogen bonding is observed in the gorge of TcAChE;
this may be due to the large re®nement uncertainties, but could re¯ect the absence of this type of
interaction.
Voids in the X-ray model of 2ACE
Poorly coordinated water molecules with very
high B values are observed in all parts of the
active-site gorge. Several of the re®ned water molecules have severely unsatis®ed hydrogen bond
capacities, i.e. they have far fewer than the ideal
number of four hydrogen bond partners in their
close vicinity. Furthermore, it seems strange that
there are distinct water clusters with distances of
Ê , a distance just too long to permit forabout 4 A
mation of a decent hydrogen bond. It is hard to
decide whether these distances are real, or whether
they re¯ect de®ciencies of the structure models.
If not all water sites have been located in 2ACE,
this would result in voids in the structure, as well
as in poor coordination of neighboring located
water sites. To check for the presence of such voids
in the water-®lled gorge of 2ACE, we used the program SURFNET (Laskowski, 1995), with a miniÊ . By far the largest
mum sphere radius of 1.3 A
Ê 3) is found near the gorge entrance, adjavoid (67 A
cent to the side-chain indole group of Trp279.
Because in 1VOT, 2ACK and 1VXR, this gap is
found to be ®lled with three or four water molecules, we assume that in native TcAChE, too, it is
®lled with water molecules which are not resolved
Ê 3) occurs in
in 2ACE. The second largest gap (21 A
the central part of the gorge, with short contacts to
Ê ), Wat749 (3.2 A
Ê ) and Wat633
Wat696 (2.4 A
Ê
(3.5 A). The two next largest gaps are already
Ê 3) is located between the
much smaller. One (10 A
Table 8. The putative aromatic hydrogen bond Trp279Ne1-H Tyr70p
Ê)
N C range (A
Ê)
N M (A
Ê)
H C range (A
Ê)
H M (A
N-H M ( )
Closest atom ˆ X
N-H X ( )
M, aromatic midpoint.
2ACE
1VOT
2ACK
1EVE
1VXR
3.8-4.4
3.9
2.8-3.7
3.0
146
Cg
170
3.8-4.4
4.0
2.9-4.0
3.2
138
Cd1
152
3.6-4.2
3.7
2.6-3.6
2.8
141
Cg
167
3.5-4.4
3.9
2.5-4.0
3.0
138
Cd1
163
3.5-4.6
3.8
2.5-3.9
3.0
138
Cd1
153
726
Water Molecules in Acetylcholinesterase
Figure 7. (Legend opposite)
Water Molecules in Acetylcholinesterase
727
Figure 7. Representations of the
putative hydrogen bond interactions within the active-site gorge
for native TcAChE (2ACE) and for
four inhibitor complexes. The gorge
lining is drawn schematically, with
main-chain groups shown as white
boxes, and side-chains as shaded
boxes. (a) 2ACE; (b) 1VOT; (c)
2ACK; (d) 1EVE; (e) 1VXR. ConÊ are shown. The color
tacts < 3.5 A
coding identi®es different water
groupings assigned in the gorge of
2ACE, related to the coloring of
water molecules in Figure 6. In (b)
to (e), water molecules that are not
seen in 2ACE are drawn in pink.
Wat1249 in 1EVE is located outside
the gorge.
oxyanion hole and the acyl pocket, with short disÊ ), Wat742 (3.1 A
Ê ), Ser200Og
tances to Wat682 (2.8 A
Ê
Ê
Ê ), Phe331p
(3.2 A), Phe288p (4.2 A), Phe290p (4.8 A
Ê ) and Trp233p (4.8 A
Ê ). A second 10 A
Ê 3 gap is
(4.5 A
located in the choline-binding site, with short disÊ ), His440O
Ê ), Trp84Ne (3.1 A
tances to Wat679 (2.9 A
Ê
Ê
Ê ). The
(3.2 A), Trp84p (3.9 A) and Phe330p (4.6 A
latter two gaps are too small to accommodate
water molecules, suggesting that the hydrophobic
choline and acyl-binding sites are actually poorly
populated with water. Further small gaps are
found in most parts of the gorge, as has been
shown for the E2020-TcAChE complex (Kryger
et al., 1999).
(ÿ)-Huperzine A-TcAChE complex (1VOT)
The herbal alkaloid, (ÿ)-huperzine A (see
Figure 8(a)), is a reversible inhibitor of AChE that
has found use in Chinese folk medicine (Liu et al.,
1986; Kozikowski et al., 1992; Kozikowski &
TuÈckmantel, 1999). The polar contacts in the gorge
for the (ÿ)-huperzine A-TcAChE complex (Raves
et al., 1997), are shown in Figure 7(b). The inhibitor
molecule displaces all seven water molecules in the
lower part of the gorge that are not positioned in
pockets. The two pocket water molecules, equivalent to Wat615 and Wat725 in 2ACE, are conserved.
The water molecule in the oxyanion hole is shifted
Ê compared to that in 2ACE, so that
by about 2.1 A
the polar contacts to Gly118N and Ala201N are
lost, whereas a possible interaction with the p-electrons of Trp233 is discerned (the distances to
Ê , respectTrp233Cz3 and Trp233CZ are 3.2 and 3.3 A
ively). The carbonyl group of the inhibitor is
roughly at the position of Wat6082ACE, and the
ethyl group forms a short CH3 O contact with
His440O. Although the C O contact is only
Ê , the C-C O angle of 167 is not indicative
3.0 A
of appreciable hydrogen bonding (Desiraju &
Steiner, 1999). The primary amino group of the
inhibitor, which is presumably charged, does not
occupy a position equivalent to a water site in
2ACE. It interacts with a water molecule in the
upper part of the gorge, and with the aromatic
faces of Phe330 and Trp84 of the choline-binding
Ê to Phe330, and
site (distances to midpoint, 4.8 A
Ê
4.3 A to the ®ve-membered ring of Trp84). Overall,
the inhibitor makes surprisingly few speci®c interactions with the enzyme. Possibly, binding to the
enzyme is ef®cient because the inhibitor so neatly
displaces a pre-existing pattern of water molecules,
with a 3D shape resembling it, in the lower part of
the gorge (Livnah et al., 1993; Raves et al., 1997).
In the upper part of the gorge, the four-water
array shown in green in Figure 7 is well conserved
and, again, has no polar contact with the other
water arrays. Of the other six water molecules
located in 2ACE, ®ve are found in similar positions
728
Water Molecules in Acetylcholinesterase
Figure 8. Chemical structures of
the AChE inhibitors referred to in
this study: (a) (ÿ)-huperzine A; (b)
edrophonium; (c) E2020; (d) nonaged conjugate with the nerve
agent VX; the side-chain and Ca of
Ser200 are also shown.
in 1VOT, one is missing, and three additional sites
are found populated.
Edrophonium-TcAChE complex (2ACK)
Edrophonium (see Figure 8(b)) is a competitive
inhibitor of AChE that is used clinically to diagnose myasthenia gravis (Hobbiger, 1976). The
polar contacts in the edrophonium-TcAChE complex (Harel et al., 1993; Ravelli et al., 1998) are
shown in Figure 7(c). The small inhibitor molecule
displaces only four water molecules in the lower
part of the gorge. The other water molecules are
all located in positions very similar to those in
2ACE. The hydroxyl group of the inhibitor is
inserted between the side-chains of Ser200 and
His440, thereby disrupting the characteristic
hydrogen bond scheme of the catalytic triad,
and
forming
a
hydrogen-bonded
chain,
Ser200Og OHEdr His440Ne2 (Harel et al.,
1993). The water molecule in the oxyanion
hole may interact with the p-electron cloud of
Trp233 similarly to the equivalent water in the
(ÿ)-huperzine A complex. The quaternary
ammonium group of the inhibitor is located in
the choline-binding site, where it forms a cationp interaction with Trp84.
In the upper part of the gorge, the location of the
water molecules is very similar to that in 1VOT,
except that an additional water site, Wat831,
bridges the water arrays in the upper and lower
parts of the gorge. The four water molecules
shown in green are not in polar contact with the
others.
E2020-TcAChE complex (1EVE)
E2020 (donepezil, Aricept1; see Figure 8(c)) is a
reversible inhibitor of AChE that is used for symptomatic treatment of Alzheimer's disease
(Kawakami et al., 1996; Nightingale, 1997). In its
complex with TcAChE, the elongated inhibitor
molecule stretches along the gorge axis, reaching
from the bottom to the gorge opening (Kryger et al.,
1999). Despite the bulk of the inhibitor, 16 water
molecules are still seen in the active-site gorge
(Figure 7(d)). In the lower part of the gorge, the
benzyl group and the piperidinium group displace
only two of the water molecules found in 2ACE.
The eight remaining water molecules in this region
Water Molecules in Acetylcholinesterase
are all in positions similar to those of their equivalents in 2ACE. Two of them make polar interactions with the E2020 molecule, one forms an N‡H OW hydrogen bond with the piperidinium
group, and one a clear aromatic hydrogen bond
with the benzyl ring (Wat1160 C(Ph) ˆ 3.6Ê , Wat1160 M(Ph) ˆ 3.4 A
Ê ). The benzyl
3.8 A
group is also involved in a face-to-face stacking
interaction with Trp84, and the tertiary ammonium
group in a cation-p interaction with Phe330. Compared to 2ACE, Wat1160 (equivalent to
729
Wat7422ACE) is slightly shifted, resulting in a polar
contact with the water molecule in the oxyanion
Ê , compared to 3.8 A
Ê
hole (with a distance of 2.8 A
in 2ACE).
In the upper part of the gorge, most of the water
molecules are in positions very similar to those of
their equivalents in 2ACE, if not replaced by the
inhibitor. The six-membered ring of the indanone
group forms a face-to-face stacking interaction
with Trp279 of the peripheral binding site.
Figure 9. Arrangement of water in the active-site gorge of 1VXR adjacent to the side-cXhains of Trp84 and Trp279.
(a) Region adjacent to Trp84; (b) Region adjacent to Trp279. Shown are sections of the ®nal electron density map
(3Fo ÿ 2Fc map at 1.3 s). Note that part of the water arrangement has continuous electron density, whereas other
water molecules have isolated spherical electron densities. Wat1251 and Wat1286 are outside the gorge.
730
Non-aged VX-TcAChE conjugate (1VXR)
The organophosphorus nerve agent VX is an
irreversible inhibitor of AChE that functions by
phosphonylation of the active-site serine residue
(see Figure 8(d)) (Aldridge & Reiner, 1972). The
corresponding crystal structure has been deterÊ resolution (Millard et al., 1999b), a
mined at 2.2 A
higher resolution than for the other structures in
our sample. The covalent inhibitor displaces the
water molecule in the oxyanion hole, Wat6822ACE,
and the water closest to the acyl pocket,
Wat7422ACE (Figure 7(e)). The remaining water
arrangement is similar to that in 2ACE, but some
interesting features are visible in greater detail, in
particular at the choline-binding site and the
peripheral site. In contrast to the other water
molecules in the gorge, which typically have
clear and well-de®ned positions, the electron
density maps in these regions are more poorly
de®ned and indicative of disorder, as is shown in
Figure 9.
In the choline-binding site, an elongated cloud of
electron density is observed (Figure 9(a)). There are
four maxima that could be modeled as water molecules, and which are involved in intermolecular
contacts typical for water, but the continuous
nature of the electron density is indicative of disorder. In 2ACE, very similar continuous electron
density is observed (Wat633, Wat679, Wat678). Of
the re®ned water sites, Wat1008 is facing the sixÊ ),
membered ring of Trp84 (Wat1008 M ˆ 4.0 A
Ê
and Wat1007 is located 3.9 A from the aromatic
midpoint of Phe330 (not seen in Figure 9(a)). In the
peripheral binding site, similar continuous electron
density is found (Figure 9(b)), which extends from
the central part of the gorge up to the gorge opening. Also in this case, there are maxima that can be
re®ned as water molecules, but some disorder is to
be anticipated. It should be noted that both faces
of the indole group of Trp279 are in contact with
water molecules, buried Wat1235 (equivalent to
Wat7042ACE, Figure 4), and gorge Wat1004; the distances to the aromatic midpoint, however, are
Ê , respectively. It is
quite different, 3.1 and 3.9 A
likely that a similar water arrangement exists in
native TcAChE, but it is not seen in 2ACE, probably due to the lower resolution compared to
1VXR.
Discussion
The data presented above allow us to draw a
number of conclusions, but also raise numerous
questions. The discussion of these points parallels
their presentation in Results.
Buried water molecules
TcAChE contains a large number of buried
water molecules, about twice as many per residue
as in more typical proteins (Williams et al., 1994).
Water Molecules in Acetylcholinesterase
About half of the buried water is concentrated in
one particular region of the protein, enclosed by
long and ¯exible loops, and immediately adjacent
to the active-site gorge. In this region, water molecules are mainly aggregated in clusters, and only
a few are isolated. These water molecules are
remarkably well conserved among the ®ve crystal
structures studied. Several other buried and wellconserved water molecules are located close to the
catalytic triad, somewhat separated from the cluster region. The rest of the buried water is loosely
scattered over the protein, with no obvious connection to the active-site. The high content of buried
water suggests that it plays a functional role in the
enzyme beyond the local structure stabilization
that is usual for buried water molecules.
Buried water molecules in proteins should not
be considered as trapped for inde®nite lengths of
time. In fact, their rate of exchange with bulk solvent is rather rapid. Typical NMR measurements
give upper and lower bounds for the residence
time of about 10ÿ3 and 10ÿ8 second, respectively
(DoÈtsch & Wider, 1995; Denisov et al., 1995;
Connelly & McIntosh, 1998). For an engineered
hydrophobic cavity in phage T4 lysozyme, it was
found that even relatively large molecules, such as
benzene and indole, can enter and leave with
bimolecular rate constants of 106-107 Mÿ1 sÿ1,
within about two to three orders of magnitude of
the diffusion-controlled limit (Feher et al., 1996).
Such a fast exchange of buried water and of larger
molecules with the solvent requires large-scale
Ê ) of backbone atoms that are at
¯uctuations (1-2 A
least equally fast. High water mobility, without
obvious diffusion channels, occurs even in much
smaller and more rigid biomolecular systems, such
as hydrated b-cyclodextrin (Steiner & Koellner,
1994; Steiner et al., 1995), and does not, therefore,
come as a surprise in proteins.
Sites of buried water molecules are not always
fully populated. The presence of buried water molecules, with occupancies of 10-50 %, has been
demonstrated by NMR methods in hen egg white
lysozyme (Otting et al., 1997). Such water molecules will normally not be detectable by crystallography. In cavities with a mainly hydrophobic
surface, water molecules can be heavily disordered,
so that they also cannot be located by diffraction
methods though still visible in NMR experiments
(Ernst et al., 1995). It could be shown that even
apparently ordered buried water molecules show
pronounced orientational disorder (Denisov et al.,
1997).
All these circumstances point to a high mobility
of buried water molecules, even of those that are
observed with low temperature factors in diffraction experiments. For the most interesting buried
water molecules in TcAChE, those in the cluster
region neighboring the active-site gorge, it is
reasonable to assume that they also exchange very
rapidly, both among themselves and with the bulk
solvent. The paths along which such exchanges
occur is not obvious; it might involve the outer sur-
Water Molecules in Acetylcholinesterase
face of the protein as well as the gorge wall. There
is probably not one distinct path, but many, which
are all temporarily opened and closed by ¯uctuations in the protein conformation.
One role that can be considered for the large
arrays of buried water that we observe in the
AChE crystal structures is to act as a lubricant that
permits, or even facilitates, large-scale ¯uctuations
of the loops forming the active-site gorge. Water
molecules can stabilize a certain conformation by
formation of suitable hydrogen bonds but, due to
their high mobility, this stabilization is not rigid,
and allows motion between different low-energy
conformations. This will permit the entire region to
be ¯oppy without being really unstable. Largeamplitude ¯uctuations of at least part of the gorge
wall have been shown to be necessary to allow
traf®c of the substrate to the active-site and, presumably, also play a key role in the product clearance by routes that have yet to be established (for
a recent reference, see Bartolucci et al., 1999). The
model of buried water acting as a lubricant,
although speculative, offers an attractive explanation for the structural ¯exibility that is a prerequisite for the activity of AChE, and should thus
be further investigated.
Gorge water molecules
Our analysis of buried water molecules reveals a
high degree of conservation within the set of ®ve
Ê . The posstructures with resolutions of 2.2-2.5 A
itions and coordination geometries of these water
molecules are all chemically reasonable, and the
pattern of intermolecular interactions is typically
conserved even in details (within the standard
Ê in this resoluncertainty in distances of ca 0.2 A
ution range). This gives us the con®dence to use
similar methods to analyze the water molecules in
the active-site gorge.
The primary observation is that within the ®ve
structures, the water molecules in the gorge are
conserved in their positions if they are not displaced by a bound inhibitor. Inhibitors typically
displace a de®ned set of water molecules, whereas
the rest remain in positions similar to those that
they occupy in the native enzyme. This is true both
for the complex with the bulky E2020 molecule as
well as for the conjugate with the small VX moiety.
The hydrogen bond coordination of the gorge
water molecules is very poor, on the average, and
they form relatively few speci®c interactions with
the enclosing wall. When averaged over the whole
gorge, the number of polar contacts per water molecule is only 2.9 in 2ACE, as compared to 3.6 for
the buried water molecules, and an ideal value of 4
(as in ice). This means that the total hydrogen
bond energy per molecule is low, certainly lower
than in bulk water. The gorge water is, therefore,
energetically activated compared to bulk water.
The wall of the active-site gorge has a very irregular shape, as well as an irregular distribution of
polar and apolar moieties. There are some polar
731
pockets, which accommodate water molecules.
These are very well conserved within the set of
®ve crystal structures, as was the case for the buried water molecules. They have low displacement
parameters, B, and are typically not replaced by
bound inhibitors (Wat615, Wat725 in 2ACE). The
oxyanion hole, too, is such a polar pocket, but the
functional water molecule located there in the
native enzyme (Wat682) is displaced by some
inhibitors, in particular by those that mimic a substrate in its tetrahedral transition state.
Apart from the polar pockets, there are also
``hydrophobic'' sites, in particular the choline-binding site, the acyl pocket, and the peripheral binding
site. In the absence of inhibitors, the choline and
peripheral binding sites seem to be populated by
disordered water molecules, which are probably
involved in weak speci®c interactions with the pelectron systems of the side-chains forming these
sites. These water molecules are poorly coordinated
with hydrogen bonds, and can certainly be displaced easily by incoming inhibitors. In the acyl
pocket, there is no water molecule visible in X-ray
diffraction experiments. Acyl groups placed there
must lead to substantial energetic improvement of
the system, due to the speci®c interactions with the
aromatic rings, without any loss of corresponding
interactions by water molecules that are displaced.
The poor hydrogen bond coordination of the
gorge water, and the small number of speci®c interactions with the gorge wall, are necessarily associated with a relatively low ef®ciency of space ®lling,
e.g. with the occurrence of many voids. These voids
are typically too small to accommodate solvent molecules, but because they are many (see Kryger et al.,
1999, for the example of E2020-TcAChE), they comprise a relatively large total volume. This may be
the main reason why such a large molecule as
E2020 displaces so few water molecules upon binding. Actually, one can think of ef®cient occupation
of voids being a major driving force for ligand binding. Thus a particular inhibitor might displace a set
of water molecules in the active-site by mimicking
their geometry and speci®c interactions, and, in
addition, might ®ll voids left by the relatively
loosely packed water molecules. Ef®ciency of ligand
binding would, accordingly, be associated in part
with ef®ciency of ®lling of voids present in the
native enzyme. For TcAChE, in particular, the acyl
pocket is a region that is extremely inef®ciently
®lled with water in the native enzyme, whereas in
the inhibitor complexes studied, it is occupied by
parts of the bound ligand.
Aromatic side-chains account for a large fraction
of the surface of the active-site gorge. In the native
enzyme, relatively few of these aromatic groups
seem to interact speci®cally with water molecules,
e.g. Trp279, whereas for most, the observed water
sites are relatively distant. This is not as might be
expected, since water-aromatic interactions with
energies of ca 2.0 kcal/mol can, in principle, occur
readily (Desiraju & Steiner, 1999). The aromatic
residues of the gorge wall play a key role in ligand
732
recognition and binding through cation-p interactions. Possibly, the numerous aromatic groups
act also as a series of temporary binding sites, facilitating easy traf®c of a quaternary ligand down the
gorge (Sussman et al., 1991). Their poor interaction
with water molecules may help to explain the easy
displacement of gorge water by substrates or
inhibitors. In comparing interactions of cations and
of water molecules with aromatic groups, it must
be kept in mind that the binding energies for the
former are much larger (>10 kcal/mol, Ma &
Dougherty, 1997; also see Verdonk et al., 1993). In
a competitive situation, a con®guration associated
with cation-p interactions will, therefore, be
strongly favoured over one with water-p hydrogen
bonds.
The present study also has implications for
theoretical studies of ligand binding and for molecular dynamics simulations of protein dynamics.
In both cases, water molecules play essential roles.
Both ligand binding and ligand traf®c within the
active-site gorge of AChE most certainly cannot be
understood without taking the water molecules
into account. Even when a bulky inhibitor, such as
E2020, is bound, there are still a large number of
water molecules left in the gorge, which cannot be
neglected. Similarly, one cannot hope to gain a realistic picture of the protein dynamics of AChE
without fully considering the roles of the buried
water molecules, which also play an integral role
in stabilizing the fold of the protein.
Finally, we wish to make a methodological comment. We believe that we have shown that elaborate analysis of hydration patterns is possible from
Ê.
protein structures of medium resolution, 2.2-2.5 A
We are in agreement with the commonly held
view that such an analysis is questionable for individual crystal structures in this resolution range. If,
however, a relatively large set of closely related
but independently re®ned structures is available,
water sites that are conserved within the set can be
analyzed reliably.
Methodology
Identification of buried water molecules
Buried water molecules in proteins are those that are
structurally isolated from the bulk solvent. For water
molecules located deep inside a protein, this is a straightforward de®nition, and they can easily be identi®ed by
simple inspection of a crystal structure. However, there
are water molecules, in surface clefts or pockets, for
which it is not possible to decide unambiguously
whether they are in contact with the bulk solvent. For
such borderline cases, different methods for identi®cation of buried water molecules can yield different
results, and considerable caution in interpretation is
advisable.
Several algorithms have been proposed for identi®cation of buried water molecules in protein crystal
structures. Most of the earlier studies follow the
methods of Lee & Richards (1971) and of Connolly
(1983), which are based on calculation of solvent-
Water Molecules in Acetylcholinesterase
accessible surfaces. In this approach, a buried water
molecule is one that is encapsulated by an internal
surface (e.g. used by Rashin et al., 1986; Sreenivasan
& Axelsen, 1992). In a recent alternative method proposed by Williams et al. (1994), a buried water molecule is de®ned as one that cannot be connected by a
continuous series of water-water hydrogen bonds with
the bulk solvent. In practice, this can be explored by
using the program PRO ACT (Williams et al., 1994).
The program ®lls up the protein surface that is not
covered by experimental water molecules with theoretical water sites. Experimental water molecules that
are then not connected with the bulk solvent by polar
contacts mediated by experimental or theoretical water
molecules are classi®ed as buried. In our analysis, we
used this latter method to identify buried water molecules in TcAChE structures. We used the default parameters of PRO ACT, which assigns the water
Ê , and a
molecule a characteristic polar radius of 1.50 A
Ê . Other polar groups
maximum polar radius of 1.63 A
are assigned different polar radii, based on earlier
statistical studies of water-protein distances (Thanki
et al., 1988; Walshaw & Goodfellow, 1993). Only
water-water contacts shorter than twice the sum of
Ê ) are considered as
the maximum polar radius (3.26 A
possible hydrogen bonds.
Following the automated analysis with PRO ACT, the
water molecules labeled as buried were individually
inspected on a graphics display (program Insight-II), and
their intermolecular contacts were examined to a maxiÊ . Because we prefer to consider
mum distance of 4.0 A
Ê as potentially hydrogen
water-water contacts up to 3.5 A
bonded (see below), several water molecules labeled as
buried by the program were not considered so by us.
Furthermore, we did not consider as buried water molecules that are trapped in the active-site gorge by a
bound inhibitor. Although not in direct contact with the
bulk solvent, such water molecules do come into contact
with it if the inhibitor is released. Finally, we note that it
was not necessary to exclude any water positions
because of unrealistic or suspicious contacts and coordination geometries.
Hydrogen bond criteria
If only O O or N O distances are known, it is not
possible, in general, to assign hydrogen bond character
unambiguously to a given contact. Only for short disÊ is assignment as a hydrogen
tances of O/N O < 3.0 A
bond quite reliable. For longer distances, a contact can,
but need not be, associated with a hydrogen bond, and
the corresponding probability falls off with increasing
distance. In consequence, selecting a distance cutoff
value is dif®cult. Exclusive criteria such as O/
Ê neglect many long hydrogen bonds
N O < 3.2 A
(Jeffrey & Saenger, 1991), whereas permissive criteria
Ê include numerous contacts that
like O/N O < 3.8 A
are not, in fact, associated with a hydrogen bond (Steiner
& Saenger, 1994). As a compromise value, we consider
Ê as
here polar contacts shorter than O O/N ˆ 3.5 A
suggestive of hydrogen bonding, or as putative hydrogen bonds. For hydrogen bonds formed by N-H donors
with geometrically de®ned H-atom positions, we
additionally require that H O distances be shorter
Ê , and N-H O angles be larger than 90 .
than 3.0 A
Weak polar interactions of water molecules with C-H
groups were not systematically analyzed at this stage,
but will be the subject of a subsequent study.
Water Molecules in Acetylcholinesterase
Structural data set
To ®nd a structural sample suitable for detailed analysis of water molecule positions, we inspected ten crystal
Ê.
structures of TcAChE in the resolution range 2.2-3.0 A
In Table 1, both the number of experimental water molecules, and the number of buried water molecules are
given. The number of buried water molecules increases
Ê , at which resolwith improving resolution up to 2.5 A
ution saturation seems to be reached. Notably, the three
Ê (2ACE, 1VOT and 1EVE)
structures determined at 2.5 A
differ by a factor of almost 2 in the total number of
water molecules, but the number of buried water molecules differs by only one molecule. This suggests that
Ê is fairly
the set of buried water molecules found at 2.5 A
complete, whereas at lower resolutions it is not. In consequence, the analysis here utilizes the ®ve TcAChE crystal
Ê . Further relstructures with resolutions of at least 2.5 A
evant information concerning these structures is given in
Table 1, and the chemical formulae of the inhibitors are
shown in Figure 8. It has been shown above that, within
this set, the positions of most of the buried waters are
very well conserved, justifying the selection made.
The ®ve structures analyzed fall into the relatively
Ê . A realistic estimate
narrow resolution range of 2.2-2.5 A
for the standard uncertainty of interatomic distances
Ê . This
involving water molecules might be 0.15-0.25 A
allows de®nition of hydrogen bond con®gurations with
reasonable reliability, but numerical values for distances
must be viewed with caution.
Acknowledgments
This work was supported by the Minerva Foundation
(G. K. and T. S.) Munich, the U.S. Army Medical and
Material Command (contracts DAMD17-97-2-7022), the
U.S. Army Scientist/Engineer Exchange Program, the
EU 4th Framework Program in Biotechnology, the Kimmelman Center for Biomolecular Structure and Assembly
(Rehovot), and the Dana Foundation. The generous
support of Mrs Tania Friedman is gratefully acknowledged. I.S. is the Bernstein-Mason Professor of Neurochemistry.
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Edited by R. Huber
(Received 24 September 1999; received in revised form
15 December 1999; accepted 15 December 1999)
http://www.academicpress.com/jmb
Supplementary Material comprising Tables with B
values of individual buried and gorge water molecules, polar contacts and visible water molecules
is available from JMB Online