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Quantification of CationЦ Interactions in ProteinЦLigand Complexes Crystal-Structure Analysis of Factor Xa Bound to a Quaternary Ammonium Ion Ligand.

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Molecular Recognition
Quantification of Cation?p Interactions in
Protein?Ligand Complexes: Crystal-Structure
Analysis of Factor Xa Bound to a Quaternary
Ammonium Ion Ligand**
Kaspar Schrer, Martin Morgenthaler, Ralph Paulini,
Ulrike Obst-Sander, David W. Banner,* Daniel Schlatter,
J#rg Benz, Martine Stihle, and Fran&ois Diederich*
In our molecular recognition studies, aimed at quantifying the
energetics of individual protein?ligand interactions,[1] we
became interested in exploring cation?p interactions[2, 3] in
the D-pocket of thrombin, a central serine protease in the
blood coagulation cascade. The bottom of this hydrophobic
pocket is lined by the indole residue of Trp 215 (Figure 1), an
aromatic amino acid side chain frequently involved in cation?
p interactions in biological systems.[4] To probe this interaction, we prepared the tricyclic inhibitors[5] ( )-1 and ( )-2,
predicted by computer modeling[6] to position a quaternary
ammonium ion and an uncharged tert-butyl group above the
indole ring of Trp 215.
The synthesis of ( )-1 started with the 1,3-dipolar
cycloaddition between maleimide 3, aldehyde 4, and l-proline
(5) to give ( )-6, which was transformed into amidinium salt
( )-7 using a Pinner-reaction (Scheme 1, for full experimen-
[*] Dr. U. Obst-Sander, Dr. D. W. Banner, Dr. D. Schlatter, Dr. J. Benz,
M. Stihle
Pharma Division
Pr klinische Forschung
F. Hoffmann-La Roche AG
4070 Basel (Schweiz)
Dr. K. Sch rer, M. Morgenthaler, R. Paulini, Prof. Dr. F. Diederich
Laboratorium f5r Organische Chemie
ETH H8nggerberg
HCI, 8093 Z5rich (Switzerland)
Fax: (+ 41) 1-632-1109
[**] This work was supported by a grant from the ETH Research Council
and by F. Hoffmann-La Roche, Basel. We thank Olivier Kuster, Dr.
Thomas Tschopp, and Dr. Alain Gast for the biological assays as well
as Dr. Michael Hennig for his support of this work.
Supporting information for this article is available on the WWW
under or from the author.
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200500883
Angew. Chem. Int. Ed. 2005, 44, 4400 ?4404
Table 1: Activities (inhibition constants Ki [mm]) of inhibitors ( )-1 and
( )-2.
Ki [mm]
Factor VIIa
> 69[a]
> 69[a]
Factor Xa
[a] Upper limit of the assay.
Figure 1. Schematic representation of the binding mode of the
designed inhibitor ( )-1 in the active site of thrombin. Only the
3aS,4R,8aS,8bR configured enantiomer is predicted to bind.[5] Reference inhibitor ( )-2 is predicted by computer modeling to adopt a
similar binding mode.
Scheme 1. Synthesis of inhibitor ( )-1. a) MeCN, 80 8C, 32 %;
b) 1. HCl, MeOH, CHCl3, 4 8C; 2. NH4Cl, MeOH, H2O, 65 8C, 60 %;
c) Me3N, MeOH, RT, 90 %.
tal details, see Supporting Information). Nucleophilic substitution with Me3N subsequently afforded ( )-1. The synthesis of reference compound ( )-2 followed a similar route
(see Supporting Information).
The activities of inhibitors ( )-1 and ( )-2 were tested,
as previously described,[7] towards thrombin, the digestive
enzyme trypsin, and two other serine proteases from the
blood coagulation cascade, Factor VIIa and Factor Xa
(Table 1), representing targets in the treatment of thrombotic
diseases.[8] No inhibition of Factor VIIa was observed within
the limits of the assay, and the binding of both ligands to
trypsin was of similar, moderate strength. In contrast, their
affinities against thrombin and Factor Xa differ greatly.
The onium ion inhibitor ( )-1 showed a much lower
activity (Ki = 7.76 mm) towards thrombin than the reference
Angew. Chem. Int. Ed. 2005, 44, 4400 ?4404
compound ( )-2 (Ki = 0.13 mm). According to molecular
modeling,[6] the large difference in activity (Ki(1)/Ki(2) = 60
and DDG = 2.5 kcal mol 1 in favor of the tert-butyl inhibitor)
cannot be explained by different binding geometries of the
two imide-N substituents in the hydrophobic D-pocket.[5]
Rather, the data suggest that cation?p interactions between
the onium ion of ( )-1 and a single indole residue (of Trp 215)
are not very effective and cannot compensate the costs of
desolvation of the ionic residue upon incorporation into the
D-pocket. Such desolvation costs do not occur in the
complexation of a tert-butyl residue, and ( )-2 binds with
an affinity similar to that of other inhibitors of this class, which
direct large aliphatic residues into the D-pocket.[9a]
In contrast, inhibitor ( )-1 shows good activity towards
Factor Xa (Ki = 0.28 mm) while the activity of reference
compound ( )-2 is very low (Ki = 29 mm). Thus, towards
Factor Xa, the difference in activities (Ki(2)/Ki(1) = 100,
DDG = 2.8 kcal mol 1) is even more distinctive than in
thrombine but in the opposite sense, favoring onium ion
recognition.[10] Again, modeling suggested nearly identical
binding modes for both inhibitors. To elucidate the origin of
the strong complexation of the onium ion, the X-ray crystal
structure of ( )-1, co-crystallized with Factor Xa, was
measured at a resolution of 1.64 9 (Figure 2).[11]
In the structure, the 3aS,4R,8aS,8bR configured enantiomer of ( )-1 (ent-1) adopts a binding geometry at the active
site of Factor Xa similar to that predicted for the related
serine protease thrombin.[9b] The phenylamidinium residue is
incorporated in the S1 pocket, and interacts with the
carboxylate side chain of Asp189 (d(NиииO) 3.2 and 3.3 9),
and the ?lower? imide-C=O unit undergoes hydrogen bonding to the backbone-NH group of Gly 216 (d(OиииN 3.1 9).
The tricyclic scaffold of ent-1 occupies the central cavity of
Factor Xa (Figure 2 b).
As expected from computer modeling, the quaternary
ammonium ion of ent-1 binds in the S4-pocket of Factor Xa,
which is lined by Trp 215, Tyr 99, and Phe 174. The three side
chains form an ?aromatic box?, with the onium ion located in
its center. The shortest distances between the N+ center and
closest C atoms of the surrounding aromatic rings are nearly
ideal (4.6 to 5.0 9, Figure 2 a) for strong cation?p interactions
(see below).
A survey of the literature shows that the S4-pocket of
Factor Xa has indeed been filled many times by basic residues.
Examples are 4-amino-substituted pyridines, piperazines,
pyrrolidines, amidines and phenylamidines, imidazoles and
imidazolines, and benzylamines.[12] We presume that the
positively charged residues occupy the cation binding site in
the aromatic box in their protonated form. The occupation of
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
not at the origin of the preference of the S4-pocket for
cationic residues.
From these results a new interpretation arises concerning
the special feature of this pocket to accept basic residues: in
their protonated form, these residues undergo efficient
cation?p interactions with the aromatic box formed by
Tyr 99, Trp 215, and Phe 174. The difference in binding free
enthalpy of DDG = 2.8 kcal mol 1 favoring ( )-1 over ( )-2
corresponds to an averaged value of about 0.9 kcal mol 1 per
aromatic ring. This value is in accordance with those reported
for cation?p interactions with individual aromatic rings.[14]
Despite the large free enthalpy increment for the interaction
with a single aromatic ring, the results obtained in this work
for the D-pocket of thrombin suggest that efficient cation?p
interactions require the ?solvation? of the cation by several
aromatic rings.
Inspired by these results, we performed Protein Data
Bank (PDB) searches[15] to identify similar aromatic boxes for
the complexation of ammonium ions. Figure 3 shows exam-
Figure 2. a) Binding mode of inhibitor (3aS,4R,8aS,8bR)-1 in the active
site of Factor Xa according to X-ray crystal-structure analysis. Interatomic distances are in K. Shortest distances between onium ion
methyl carbon atoms and carbon atoms of the aromatic box are 3.9 K
(to Tyr 99), 3.9 K (to Phe 174), and 3.6 K (to Trp 215); red O, blue N,
yellow S, green Cligand, gray Cprotein, b) Schematic representation of the
complex between Factor Xa and the active enantiomer of ( )-1.
the S4-pocket by an N,N,N-trimethylanilinium residue has
also been reported.[13]
In some complexes, the side chains of Glu 97 or Glu 217 in
Factor Xa undergo weak ion pairing with phenylamidinium
ions occupying the S4-pocket.[12a, 13] However, in the large
majority of cases, the two carboxylate groups are located at
positions remote from the cationic site and are oriented
towards the solvent, as seen in the complex of Factor Xa with
ent-1 (d(NиииCO2 )Glu 97 9.9 9, d(NиииCO2 )Glu 217 8.0 9). Thus,
Coulombic interactions with the two carboxylate groups are
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Examples from PDB searches showing quaternary ammonium ions bound in the S4-pocket of Factor Xa (a) and in similar aromatic boxes of other proteins (b?d). The shortest ligand?protein distances are indicated in K; red O, blue N, gray C. PDB-codes: a) 1LPK,
b) 1PFB, c) 1R9Q, d) 1LN3. See text for details.
ples for quaternary ammonium ions bound to different
proteins. Figure 3 a depicts the already mentioned trimethylanilinium residue in the S4-pocket of Factor Xa (PDB-code:
1LPK).[13] The distances of the N+ center to the three aromatic
rings are very close to those found in our study. Figure 3 b
features histone H3 permethylated at Lys 27 in the polycomb
chromodomain of Drosophila (1PFB),[16] while Figure 3 c
shows an N,N-dimethylated proline in the periplasmic ligand
binding protein ProX from E. Coli (1R9Q).[17] At least one of
the three side chains forming the aromatic boxes shown in
Figure 3 a?c is a Trp residue. The orientation of the cationic
center is always close to the normals passing through the
centroids of the aromatic planes which provides for optimal
interactions with the p systems. Figure 3 d shows a box of four
aromatic side-chains from human phosphatidylcholine transfer protein complexed with the choline moiety of its substrate
Angew. Chem. Int. Ed. 2005, 44, 4400 ?4404
(1LN3).[18] Similar to the previous structures, the cationic
center is located near the centroid normals of the p systems of
Trp 101 and Tyr 155. In contrast, the ring planes of Tyr 114 and
Tyr 116 are slightly twisted. Their normals are not oriented
perfectly towards the cation. Apparently, in this example, the
loss of interactions caused by the twisted orientation of these
side-chains is compensated by the presence of a fourth
aromatic residue. In all the structures shown in Figure 3 the
shortest distances between the N+ center and the aromatic
side chains are between 4?5 9 (d(N+иииCarom)), which seems to
be the optimal separation for quaternary ammonium ion?p
interactions. For further examples of quaternary ammonium
ions in aromatic boxes, see Figures 5?9 in the Supporting
The PDB search was extended to protonated tertiary
ammonium ions complexed in aromatic boxes within proteins
(Figure 4). Again, the cations are close to the centroid
Cation?p interactions are also seen in crystal packings of
small molecules, although steric factors and the requirements
of a dense crystal packing in these cases prevent the formation
of aromatic boxes. A search of the Cambridge Structural
Database (CSD) for close contacts between tetraalkylammonium ions and arene units furnished 22 examples, seven of
which displayed a second close contact to another aromatic
residue.[23] As in the case of the protein examples, cations
were located close to the centroid normals of the arene
systems. A selection of examples is included in Figure 10 in
the Supporting Information.
In conclusion, we have shown by biological assays and
protein crystallography that the aromatic box formed by the
side chains of Phe 174, Tyr 99, and Trp 215 in the S4-pocket of
Factor Xa is a very effective onium binding site. By comparing
the affinity of the quaternary ammonium ion receptor ( )-1
to that of the tert-butyl derivative ( )-2, the free enthalpy
increment for cation?p interactions in this box was determined as DDG = 2.8 kcal mol 1. PDB searches revealed the
more general occurrence of similar cation binding sites in
biological systems; hence the observations made in this
investigation should provide useful guidance in lead design
and optimization.
Received: March 9, 2005
Published online: June 13, 2005
Keywords: cation?pi interactions и Factor Xa и inhibitors и
molecular recognition и thrombin
Figure 4. Examples from PDB searches showing protonated tertiary
ammonium ions bound in aromatic boxes. The shortest ligand?protein
distances are indicated in K; red O, blue N, gray C. PDB-codes:
a) 1H37, b) 1KNA, c) 1Q0Y, d) 1UW6.
normals of the aromatic rings. An additional interesting
feature is the presence of a hydrogen bond formed on the
open side of the aromatic box between R3NH+ and an
acceptor residue of the protein, which results in additional
stabilization. This hydrogen bonding is sometimes mediated
by a water molecule. Examples are the structure of a
(protonated) tertiary amine inhibitor bound to oxidosqualene
cyclase (Figure 4 a; 1H37),[19] the complex of a protonated
N,N-dimethylated lysine residue of a histone in the HP1
chromodomain of Drosophila (Figure 4 b; 1KNA),[20] the
complex of morphine in the antibody 9B1 (Figure 4 c;
1Q0Y),[21] and the complex of nicotine in a nicotinic ACh
receptor (Figure 4 d; 1UW6).[22] Similarly, some of the protonated onium ions in the S4-pocket of Factor Xa not only
interact with the aromatic box but also form hydrogen bonds
to neighboring polar residues or H2O molecules.[12a,f] Interestingly, protonated secondary ammonium ions are not found
in such arrangements, presumably because of high desolvation costs.
Angew. Chem. Int. Ed. 2005, 44, 4400 ?4404
[1] F. Hof, F. Diederich, Chem. Commun. 2004, 477 ? 480.
[2] a) N. Zacharias, D. A. Dougherty, Trends Pharmacol. Sci. 2002,
23, 281 ? 287; b) J. C. Ma, D. A. Dougherty, Chem. Rev. 1997, 97,
1303 ? 1324; c) D. A. Dougherty, Science 1996, 271, 163 ? 168;
d) D. A. Dougherty, D. A. Stauffer, Science 1990, 250, 1558 ?
[3] For a recent review, see: E. A. Meyer, R. K. Castellano, F.
Diederich, Angew. Chem. 2003, 115, 1244 ? 1287; Angew. Chem.
Int. Ed. 2003, 42, 1210 ? 1250.
[4] P. Gallivan, D. A. Dougherty, Proc. Natl. Acad. Sci. USA 1999,
96, 9459 ? 9464.
[5] Members of this class of inhibitors show nearly identical binding
geometries in the structurally well-defined active site of
thrombin; see: a) K. SchNrer, M. Morgenthaler, P. Seiler, F.
Diederich, Helv. Chim. Acta 2004, 87, 2517 ? 2538; b) J. A. Olsen,
D. W. Banner, P. Seiler, U. Obst-Sander, A. DOArcy, M. Stihle, K.
MPller, F. Diederich, Angew. Chem. 2003, 115, 2611 ? 2615;
Angew. Chem. Int. Ed. 2003, 42, 2507 ? 2511; c) U. Obst, D. W.
Banner, L. Weber, F. Diederich, Chem. Biol. 1997, 4, 287 ? 295.
[6] a) P. R. Gerber, K. MPller, J. Comput.-Aided Mol. Des. 1995, 9,
252 ? 268; b) Gerber Molecular Design (
[7] For thrombin: K. Hilpert, J. Ackermann, D. W. Banner, A. Gast,
K. Gubernator, P. Hadvary, L. Labler, K. MPller, G. Schmid,
T. B. Tschopp, H. van de Waterbeemd, J. Med. Chem. 1994, 37,
3889 ? 3901. For Factor Xa: Kinetic enzyme inhibition test
exposing a chromogenic substrate (S) (S-2222 Km = 316 mm) at
200 mm to human Factor Xa (3 nm) in microtiter plates at room
temperature in the presence of buffer pH 7.8 (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) 100 mm,
NaCl 140 mm, poly(ethylene glycol) Mw = 6000 (PEG 6000)
0.1 %, Tween 80 0.2 %), or various concentrations of inhibitor.
The Ki-value is calculated [Ki = IC50/(1+(S/Km))] from the IC50
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
determined graphically from a dose-response curve of the
E. W. Davie, K. Fujikawa, W. Kisiel, Biochemistry 1991, 30,
10 363 ? 10 370.
a) U. Obst, P. Betschmann, C. Lerner, P. Seiler, F. Diederich, V.
Gramlich, L. Weber, D. W. Banner, P. SchRnholzer, Helv. Chim.
Acta 2000, 83, 855 ? 909; b) Only the enantiomer ent-1 which has
the same absolute configuration required for thrombin binding is
bound by Factor Xa. For a study of enantiomerically pure
tricyclic thrombin inhibitors, see refs. [5a] and [5c].
D. Monnaie, D. Arosio, N. Griffon, T. Rose, A. R. Rezaie, E.
Di Cera, Biochemistry 2000, 39, 5349 ? 5354.
For protein expression and purification, see the Supporting
Information. Crystallization and structure determination: A
solution of short-form Factor Xa (15 mg mL 1) was incubated
overnight with ligand (3.8 mm) prior to crystallization. The
protein?ligand complex was mixed with an equal volume of
crystallization buffer (0.2 m NH4OAc, 0.1m 2-[2,2?-bis(hydroxylethyl)amino]-2-hydroxymethyl-1,3-propanediol(Bis-Tris) pH 5.5,
and 25 % PEG 3350) in microbatch mode. Crystals appeared
after a week and grew to full size within two weeks. Before data
collection, crystals were transferred to crystallization buffer
supplemented with 20 % glycerol and flash-frozen in liquid N2.
Diffraction data were measured on a Bruker FR591 X-ray
generator, with 0.2 mm focus, run at 50 kV/60 mA and equipped
with an Osmic focusing monochromator and an Oxford Cryostream cooler run at 100 K. The Marresearch345 (dtb) image
plate detector was placed 110 mm from the crystal and scanned
with 0.15 mm pixel size. Exposure times were 600 s for 0.58
frames. Data from 262 frames were processed to 1.62 9
resolution using XDS (W. Kabsch, J. Appl. Crystallogr. 1993,
26, 795 ? 800). Space group P212121; unit cell dimensions a =
48.84, b = 71.67, and c = 72.75 9. For 163 433 measured reflections 32 725 were independent, R factor 6.1 % (37.4 % in the
outermost shell, 1.62?1.71 9), with completeness 98.7 %
(97.1 %) and I/s 15.5 (4.3). Data reduction used the CCP4
package (?The CCP4 Suite: Programs for Protein Crystallography? Acta Crystallogr. Sect. D 1994, 50, 760?763). The
structure was solved by molecular replacement using 1c5m.pdb
as model. Model building with Moloc[6] and refinement with
Refmac5 (G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta
Crystallogr. Sect. D 1997, 53, 240 ? 255) to 1.64 9 resolution gave
final overall crystallographic R factors of 20.4 % (observed) and
23.7 % (total), with values in the outer shell (1.64?1.68 9) of
28.6 % and 29.7 %, respectively, for 2421 atoms, including one
Na+ ion and 276 H2O molecules. The inhibitor density is very
clear. Coordinates have been deposited with the Protein Data
Bank, PDB-code: 2bok.
For selected crystal structures, see: a) S. Maignan, J.-P. Guilloteau, Y. M. Choi-Sledeski, M. R. Becker, W. R. Ewing, H. W.
Pauls, A. P. Spada, V. Mikol, J. Med. Chem. 2003, 46, 685 ? 690
(PDB-codes: 1NFU, 1NFW, 1NFX, 1NFY); b) H. Nishida, Y.
Miyazaki, T. Mukaihira, F. Saitoh, M. Fukui, K. Harada, M. Itoh,
A. Muraoka, T. Matsusue, A. Okamoto, Y. Hosaka, M.
Matsumotu, S. Ohnishi, H. Mochizuki, Chem. Pharm. Bull.
2002, 50, 1187 ? 1194 (PDB-codes: 1IQE, 1IQF, 1IQG, 1IQH,
1IQI, 1IQJ, 1IQK, 1IQL, 1IQM, 1IQN); c) S. Maignan, J.-P.
Guilloteau, S. Pouzieux, Y. M. Choi-Sledeski, M. R. Becker, S. I.
Klein, W. R. Ewing, H. W. Pauls, A. P. Spada, V. Mikol, J. Med.
Chem. 2000, 43, 3226 ? 3232 (PDB-codes: 1F0R, 1F0S, 1EZQ);
d) M. Adler, D. D. Davey, G. B. Phillips, S.-H. Kim, J. Jancarik,
G. Rumennik, D. R. Light, M. Whitlow, Biochemistry 2000, 39,
12 534 ? 12 542 (PDB-code 1FJS); e) K. Kamata, H. Kawamoto,
T. Honma, T. Iwama, S.-H. Kim, Proc. Natl. Acad. Sci. USA
1998, 95, 6630 ? 6635 (PDB-code: 1XKA); f) H. Brandstetter, A.
KPhne, W. Bode, R. Huber, W. von der Saal, K. Wirthensohn,
R. A. Engh, J. Biol. Chem. 1996, 271, 29 988 ? 29 992 (PDB-code:
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1FAX); g) A positively charged residue placed in the D-pocket
(thrombin) or S4-pocket (Factor Xa) of the enzymes was
predicted to increase inhibitor selectivity for Factor Xa over
thrombin in a computational GRID/CPCA analysis of the
corresponding enzyme active sites. However, in contrast to the
results of the computational study, the binding data for ( )-2
suggest that the analogous introduction of uncharged hydrophobic groups leads to selective inhibitors of thrombin rather
than Factor Xa; M. A. Kastenholz, M. Pastor, G. Cruciani, T.
Fox, J. Med. Chem. 2000, 43, 3033 ? 3044.
H. Matter, E. Defossa, U. Heinelt, P.-M. Blohm, D. Schneider, A.
MPller, S. Herok, H. Schreuder, A. Liesum, V. Brachvogel, P.
LRnze, A. Walser, F. Al-Obeidi, P. Wildgoose, J. Med. Chem.
2002, 45, 2749 ? 2769 (PDB-codes: 1LPG, 1LPK).
For the energetics of cation?p interactions, see: a) refs. [2?4];
b) D. L. Beene, G. S. Brandt, W. Zhong, N. M. Zacharias, H. A.
Lester, D. A. Dougherty, Biochemistry 2002, 41, 10 262 ? 10 269.
a) M. Hendlich, A. Bergner, J. GPnther, G. Klebe, J. Mol. Biol.
2003, 326, 607 ? 620; b) J. GPnther, A. Bergner, M. Hendlich, G.
Klebe, J. Mol. Biol. 2003, 326, 621 ? 636. PDB searches were
performed using Relibase 1.2.1 (March 2003). All database
entries containing ligands possessing quaternary ammonium
ions (263 structures), tertiary (1024 structures), and secondary
(1594 structures) amines were retrieved together with their
binding sites and examined by visual inspection. Molecular
graphics images were produced using the UCSF Chimera
package from the Computer Graphics Laboratory, University
of California, San Francisco (supported by NIH P41RR-01081). E. F. Pettersen, T. D. Goddard,
C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E.
Ferrin, J. Comput. Chem. 2004, 25, 1605 ? 1612.
J. Min, Y. Zhang, R.-M. Xu, Genes Dev. 2003, 17, 1823 ? 1828.
PDB-code: 1PFB.
A. Schiefner, J. Breed, L. BRsser, S. Kneipl, J. Gade, G.
Holtmann, K. Diederichs, W. Welte, E. Bremer, J. Biol. Chem.
2004, 279, 5588 ? 5596. PDB-code: 1R9Q.
S. L. Roderick, W. W. Chan, D. S. Agate, L. R. Olsen, M. W.
Vetting, K. R. Rajashankar, D. E. Cohen, Nat. Struct. Biol. 2002,
9, 507 ? 511. PDB-code: 1LN3.
A. Lenhart, D. J. Reinert, J. D. Aebi, H. Dehmlow, O. H.
Morand, G. E. Schulz, J. Med. Chem. 2003, 46, 2083 ? 2092.
PDB-code: 1H37.
S. A. Jacobs, S. Khorasanizadeh, Science 2002, 295, 2080 ? 2083.
PDB-code: 1KNA.
E. Pozharski, M. A. Wilson, A. Hewagama, A. B. Shanafelt, G.
Petsko, D. Ringe, J. Mol. Biol. 2004, 337, 691 ? 697. PDB-code:
P. H. N. Celie, S. E. van Rossum-Fikkert, W. J. van Dijk, K.
Brejc, A. B. Smit, T. K. Sixma, Neuron 2004, 41, 907 ? 914.
PDB-code: 1UW6.
F. H. Allen, Acta Crystallogr. Sect. B 2002, 58, 380 ? 388. The
CSD search was performed using a distance constraint for the
distance d between ammonium N atom and the centroid of the
aromatic ring of 3.5 9 < d < 5.5 9. Hits were limited to structures containing no errors or partial disorders, excluding
polymeric, powder, or organometallic compounds.
Angew. Chem. Int. Ed. 2005, 44, 4400 ?4404
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