Crystal structures of two vancomycin complexes with phosphate and N-AcetylD-Ala. structural comparison between low-affinity and high-affinity ligand complexes of vancomycin

Article abstract of DOI:10.1246/bcsj.20090326

Crystal structures of two vancomycin complexes with phosphate and N-acetylD-Ala (AcDA) were determined. Each complex involves two crystallographically independent vancomycin molecules (V1 and V2) in the asymmetric unit, which form a usually observed back-to-back arranged vancomycin dimer V1V2 with two disaccharide chains packed in a head-to-head manner, but only one of the two ligand-binding sites is occupied. Comparison of the published crystal structures of low-affinity (small in molecular size) ligand complexes of vancomycin with high-affinity (large) ligand complexes reveals that when the high-affinity ligand binds, three structural factors (hydrogen-bonding interactions between the two peptide-backbones and hydrophobic intra-dimer sugarring and ring (face)ring (edge) interactions) work to enhance the stabilization of the back-to-back dimer-interface, an important factor that is believed to promote antibacterial activity. It has also been revealed, by examining the high-affinity ligand complexes (including N-acetylDAlaD-Ala), that sugarligand interaction could cause different affinities of the two halves of the dimer; this is a factor responsible for the failure of the ligand binding to V1 in the AcDA complex. Possible scenarios for the formation of vancomycin complexes with low-affinity as well as high-affinity ligands are presented.

Full text of DOI:10.1246/bcsj.20090326

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 4, 391–400 (2010)
391
Crystal Structures of Two Vancomycin Complexes with Phosphate
and N-Acetyl-D-Ala. Structural Comparison between Low-Affinity
and High-Affinity Ligand Complexes of Vancomycin
Takanori Kikuchi,1 Shyam Karki,1 Ikuhide Fujisawa,1 Yoshitaka Matsushima,2
Yasushi Nitanai,3 and Katsuyuki Aoki*1
1Department of Materials Science, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441-8580
2Department of Chemistry, Hamamatsu University School of Medicine, Handayama, Hamamatsu 431-3192
3Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-gun, Hyogo 679-5148
Received December 9, 2009; E-mail: kaoki@tutms.tut.ac.jp
Crystal structures of two vancomycin complexes with phosphate and N-acetyl-D-Ala (AcDA) were determined.
Each complex involves two crystallographically independent vancomycin molecules (V1 and V2) in the asymmetric unit,
which form a usually observed back-to-back arranged vancomycin dimer V1-V2 with two disaccharide chains packed in
a head-to-head manner, but only one of the two ligand-binding sites is occupied. Comparison of the published crystal
structures of low-affinity (small in molecular size) ligand complexes of vancomycin with high-affinity (large) ligand
complexes reveals that when the high-affinity ligand binds, three structural factors (hydrogen-bonding interactions
between the two peptide-backbones and hydrophobic intra-dimer sugar-ring and ring (face)-ring (edge) interactions)
work to enhance the stabilization of the back-to-back dimer-interface, an important factor that is believed to promote
antibacterial activity. It has also been revealed, by examining the high-affinity ligand complexes (including N-acetyl-D-
Ala-D-Ala), that sugar-ligand interaction could cause different affinities of the two halves of the dimer; this is a factor
responsible for the failure of the ligand binding to V1 in the AcDA complex. Possible scenarios for the formation of
vancomycin complexes with low-affinity as well as high-affinity ligands are presented.
Vancomycin (Figure 1), which belongs to a family of
glycopeptide antibiotics that are active against gram-positive
bacteria, is clinically of special importance because it repre-
sents the last line of defense against bacteria that have
developed resistance to other antibiotics.1 However, the recent
emergence2 of vancomycin-resistant bacterial strains has
brought a limitation in the treatment of vancomycin and has
required prompt development of new therapeutic drugs.
Glycopeptide antibiotics function by binding nascent cell-wall
disaccharide-pentapeptides terminating in the sequence -D-
Ala-D-Ala to inhibit the synthesis of the linkage between
mucopeptides, thereby weakening the resulting cell-wall.3 As
the peptide-ligand recognition mechanism, the formation of
five hydrogen bonds between an antibiotic and a ligand was
proposed from spectroscopic studies4 and this has indeed
been substantiated first in crystal structures of a balhimycin
complex5 with L-Lys-D-Ala-D-Ala, or a degluco-balhimycin
complex5 with L-Ala-D-Glu-£-L-Lys-D-Ala-D-Ala and sub-
sequently in crystal structures of vancomycin complexes
with N,N¤-diacetyl-L-Lys-D-Ala-D-Ala6 and N-acetyl-D-Ala-
D-Ala.6,7
bacteria that biosynthesize such precursors.10 The drastically
reduced binding constants have been assumed11 to be due to
electro-repulsive interaction between the ester oxygen of the
C-terminal D-lactate group and the carbonyl oxygen of residue
4 of the antibiotic backbone, which forms a hydrogen bond
with the NH of the terminal D-alanine group of the normal
precursors. We have verified6 this O£O repulsion and the
concomitant lengthening of two neighboring hydrogen bonds
in the crystal structure of a vancomycin complex with N,N¤-
diacetyl-L-Lys-D-Ala-D-lactate. In this structure and also in the
N,N¤-diacetyl-L-Lys-D-Ala-D-Ala complex, of special interest
is an observation that a water molecule bridges the antibiotic
molecule to the ligand by forming hydrogen bonds with the
carboxylate terminal oxygen of antibiotic and the amide
nitrogen of the Lys residue of the ligand. We suggest6 that
this may provide a strategy for designing drugs against VRE:
the modification of the C-terminus of the antibiotic to form a
direct hydrogen bond with the amide nitrogen of the third
residue (Lys) from the C-terminus of the ligand could stabilize
the structure of the antibiotic-ligand complex more effectively.
In order to examine the significance of the water-mediated
antibiotic-ligand interaction and thus to define a minimal set of
interactions necessary for the complex formation with the
vancomycin-resistant precursors terminating in -D-Ala-D-
lactate, we have now tried the complex formation of vanco-
mycin with N-acetyl-D-Ala-D-lactate, yielding two crystalline
compounds depending on pH conditions. X-ray diffraction
On the other hand, the -D-Ala-D-Ala terminus is replaced by
-D-Ala-D-lactate in vancomycin-resistant strains (VRE)8 and
the affinity of vancomycin for precursor analogs terminating in
-D-Ala-D-lactate is decreased by a factor of the order of 1000
relative to the binding to precursors terminating in -D-Ala-D-
Ala,9 corresponding to the low activity of vancomycin against
Published on the web March 26, 2010; doi:10.1246/bcsj.20090326

392
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
Structures of Two Vancomycin Complexes
Ala-D-lactate does not bind to antibiotic, but a phosphate
buffer ion occupies the ligand-binding site (the phosphate
complex). Under slightly acidic conditions, N-acetyl-D-Ala-D-
lactate was hydrolyzed and a complex with N-acetyl-D-Ala
formed (the AcDA complex). This shows that the formation of
a stable complex with N-acetyl-D-Ala-D-lactate is considerably
difficult, suggesting that the ligand affinity of vancomycin for
N-acetyl-D-Ala-D-lactate may be little and, at least less than
that for phosphate. It may be reasonable to assume that the
stabilization by the possible hydrogen-bonding, when N-
acetyl-D-Ala-D-lactate binds to antibiotic, between the amide
nitrogen of the seventh residue of the antibiotic backbone and
the acetyl oxygen of the ligand is not enough to compensate
for electrostatic repulsion that could occur between the ester
oxygen of ligand and the carbonyl oxygen of residue 4. On the
other hand, the binding of the phosphate ligand is achieved by
the formation of a P-O-H (ligand)£O (the carbonyl oxygen of
residue 4) hydrogen bond in addition to three other conven-
tional hydrogen bonds, as described later in detail. We propose
here that since N,N¤-diacetyl-L-Lys-D-Ala-D-lactate does bind
to the antibiotic,6 the third Lys residue from the C-terminus
of the ligand might be necessary as a minimal set for the
formation of a stable complex with vancomycin-resistent
precursors terminating in -D-Ala-D-lactate, that is, the for-
mation of the water-mediated N-H (amide of the third residue
of ligand)£water£O (carboxylate oxygen of residue 7) hydro-
gen bonds6 is required for the complex formation.
Overall Structures of the Phosphate and AcDA Com-
plexes. The nomenclature is adopted by that of Loll et al.13
with modifications where necessary. In both the phosphate and
AcDA complexes, the asymmetric unit contains two vanco-
mycin molecules, referred to herein as V1 and V2. The seven
amino acid residues of vancomycin are denoted as V1:1 to
V1:7 and V2:1 to V2:7. When the backbone atoms of the
antibiotic or the ligand are mentioned, N, C¡, C, or O are used
(e.g., “V1:2 N” denotes the amide nitrogen atom of the second
residue of V1). The glucose and vancosamine sugars on each
monomer are indicated as “Glu” and “Van,” respectively. Other
atoms are referred to as the names shown in Figure 1.
Structures of the present phosphate and AcDA complexes
are isomorphous with the vancomycin complexes with
low-affinity ligands such as OAc,13,14 AcG,15 DLac,15 and
AcDLac,15 which exclusively crystallized in the tetragonal
space group P43212 and share common structural as well as
crystal packing features to one another. Hence, the asymmetric
unit contains a back-to-back arranged vancomycin dimer V1-
V2 with two disaccharide chains packed in a head-to-head
manner, and only one of the two ligand-binding pockets is
occupied by a ligand molecule in V2, as shown in Figure 2 (for
the phosphate (2a) and AcDA (2b) complexes), where the
vancosamine sugar lies over the ligand-binding pocket in V1,
while glucose locates at the corresponding position in V2.
Structures of Back-to-Back Arranged Dimers. It has
been well documented,1 based mainly on NMR studies, that
glycopeptide antibiotics have a strong propensity to form a
back-to-back-dimer through four hydrogen bonds between
two aglycons in an anti-parallel arrangement. This has
indeed been repeatedly exemplified in crystal structures of
vancomycin,6,7,12-16 aglycovancomycin,17 A-40926 aglycone,18
vancosamine
HO
(N3')
NH+3
glucose
(O3')
(C1B
)
OH
(C3')
(O4')
OH
O
(O5B
(C2')
H3C
)
O
(C5M
)
CH2OH
(C6')
O
(O5')
(O2')
CH3
(C3M
)
(O42
)
O
(C1')
(C41
(C65
)
)
(CL2)
)
Cl
(C39
)
)
(C63
O
O
(C61
)
(O66
6
4
2
(C20
(C38
)
HO
OH
O
Cl
(O59
)
)
1(C )
1
(CL6)
O
O
O
CH3
3
H
N
+
NH2
CH3
CH3
O
N
N
H
N
N
H
H
O
CONH2
H
NH
(O71
)
O
-
5
O
(O80
)
H
N
O
7
OH
H3C
-
(O53
)
HO
OH
O
(O78
)
(O79)
(OXT)
O
CH3
N-acetyl-D-Ala
Figure 1. Chemical structure of vancomycin and the bind-
ing scheme between vancomycin and a cell-wall precursor
analog, N-acetyl-D-Ala (AcDA). The amino acid residues
of vancomycin are numbered. Atoms mentioned in the text
are labeled. The hydrogen bonds between vancomycin and
AcDA are shown by broken lines.
analysis has revealed that under nearly neutral pH condition,
N-acetyl-D-Ala-D-lactate does not bind to antibiotic but instead
a phosphate buffer ion occupies the ligand-binding site. On the
other hand, under slightly acidic conditions, the hydrolysis of
N-acetyl-D-Ala-D-lactate took place to give N-acetyl-D-Ala
(AcDA) that binds to antibiotic. We report here crystal
structures of two resulting vancomycin complexes, one being
with phosphate (the phosphate complex) and the other with a
normal cell-wall precursor analog N-acetyl-D-Ala (the AcDA
complex with the space group P43212). A vancomycin-
AcDA complex with a different crystal form (triclinic P1) is
reported.12
In addition, in order to provide further information for our
better understanding of ligand-binding effects on vancomycin
structures, we here compare so far reported crystal structures
of vancomycin complexes,6,7,12-16 involving those with low-
affinity (small in molecular size) ligands such as acetate
(OAc),13,14 N-acetylglycine (AcG),15 D-lactic acid (DLac),15
and O-acetyl-D-lactic acid (AcDLac)15 and those with high-
affinity (large) ligands N-acetyl-D-Ala-D-Ala (NAAA)6,7 and
N,N¤-diacetyl-L-Lys-D-Ala-D-Ala (DALAA).6
Results and Discussion
Co-crystallization Experiments of Vancomycin with N-
Acetyl-D-Ala-D-Lactate. A total of thirty different crystal-
lization conditions were examined and two crystalline com-
pounds were obtained. X-ray diffraction analysis has revealed
that under phosphate buffer conditions (pH 6.5), N-acetyl-D-

T. Kikuchi et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
393
(a)
balhimycin,5,19 degluco-balhimycin,5 ureidobalhimycin,20 and
decaplanine,21 and this is also the case for the present
phosphate and AcDA complexes of vancomycin. Exceptions
are monomeric degradation products such as the CDP-1
analog of vancomycin22 and the CDP-1 analog of methylated
vancomycin.23
Antibiotic back-to-back arranged dimerization is believed24
to promote antibacterial action because the binding of one
monomer to the bacterial cell wall brings a second monomer
into proximity with other peptidoglycan ligands to form a
chelate with the peptidoglycan. For this action, the stabilization
of the dimer structure is of primary importance. It is also
known1 that the dimerization is cooperative with ligand
binding. We point out that the following four structural factors
could stabilize the dimer-interface: (i) hydrogen-bonding
interactions between the two peptide-backbones,1 (ii) interac-
tions between two disaccharide chains,13 (iii) hydrophobic
interactions between sugars of disaccharides and cross-linked
aromatic side chains of residues 2 or 6 across the dimer-
interface, and (iv) edge-to-face ring-ring interactions between
cross-linked aromatic side chains of residues 4 and 6.13 We here
examine which structural factors could affect the tightness
of the dimer-interface when ligand binds to antibiotic, based
on the known crystal structures of vancomycin complexes,
including the present work.
(b)
Figure 2. Stereoviews of asymmetric units in vancomycin
complexes. (a) The phosphate complex. (b) The AcDA
complex. Vancomycin molecules are represented as stick
models, and ligands are represented as ball-and-stick
models. The sugar moieties of vancomycin molecules are
represented as red sticks. Chlorine atoms or chloride ions
are drawn with green in color and sodium ions are with
violet. Red spheres represent water molecules. Hydrogen
bonds are depicted by dotted lines.
(i) Table 1 lists hydrogen-bonding distances between the two
peptide-backbones at the dimer-interface. It appears that the
distances of two chemically equivalent outer hydrogen-bonds
[V1:3 O£V2:6 N (a in Table 1) and V1:6 N£V2:3 O (d)] are
Table 1. Hydrogen-Bonding Distances (¡) at the Back-to-Back Dimer-Interface in the Vancomycin Complexes
V1:3 O£V2:6 N
(a)
V1:5 N£V2:5 O
(b)
V1:5 O£V2:5 N
(c)
V1:6 N£V2:3 O
(d)
PDB
code
CCDC
code
Ligand
Ref.
OAc
AcG
DLac
AcDLac
Phosphate
AcDA
Cu2+ ion
NAAAa)
DALAAb)
DALALacc)
3.14
3.17
3.11
3.13
3.05
3.13
2.90
3.11
3.16
3.18
2.91
2.95
2.91
2.92
2.90
2.91
2.85
2.81
2.80
2.82
2.87
2.87
2.84
2.85
2.81
2.81
2.96
2.87
2.86
2.86
3.05
3.09
3.07
3.05
3.03
3.02
3.02
3.19
3.23
3.20
1AA5
1QD8
1C0R
1C0Q
13
15
15
15
728179 this work
728180 this work
655637
693248
16
6
6
1FVM
693249
6
a) Average values are given for 3 independent V1-V2 dimers. b) Average values for 2 independent V1-V2 dimers. c) Average
values for 2 independent V1-V2 dimers.
O
Res 6
V1
Res 3
O
Res 5
H
N
NH
N
N
H
H
O
Res 4
O
b
d
c
O
Res 4
O
a
H
H
N
N
HN
N
H
V2
Res 5
O
Res 3
Res 6
O

394
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
Structures of Two Vancomycin Complexes
Figure 3. Close contacts (<4.0 ¡), drawn with dotted lines,
between two parallelly arranged disaccharide chains at the
back-to-back dimer-interface in the AcDA complex.
Figure 4. Hydrophobic contacts between sugars and aro-
matic rings at the back-to-back dimer-interface in the
AcDA complex. The sugar-ring closest contacts of two
intramolecular and two intermolecular pairs are drawn with
dotted lines.
considerably longer (weak) than those of two inner hydrogen-
bonds [a pair of V1:5 N£V2:5 O (b) and V1:5 O£V2:5 N (c)],
which fall in the normal values for N-H£O hydrogen-bonds.
When the high-affinity ligand binds, the inner hydrogen-bond b
becomes shorter, while the outer hydrogen-bond d becomes
longer. The other inner c and the other outer a hydrogen-bonds
are little influenced. Taking into consideration that the long
hydrogen-bonding (weak interaction) d may exert its minor
contribution to the structural stabilization comparing with the
shorter hydrogen-bonding b, we suspect that as a whole, the
binding of the high-affinity ligand somewhat surpasses that of
the low-affinity ligand with regard to the dimer stabilization
due to hydrogen-bonding interactions between the two peptide-
backbones.
(ii) Two parallel disaccharide chains make extensive van der
Waals contacts with each other, as shown in Figure 3 (and
Table S-1 as Supporting Information). Common to all 10
vancomycin complexes, close contacts (<4.0 ¡) occur to
similar extent between the edge (O4¤) of V1:Glu and the face
of V2:Glu, between the edge (O5B) of V2:Van and the edge (O3¤
and C3¤) of V1:Glu, and between the edge (C5M) of V2:Van and
the edge (O2¤ and C3M) of V1:Glu. Among these, very short
interatomic distances are observed between the hydroxy O4¤ of
V1:Glu and the ring atom C2¤ of V2:Glu within the range of
3.41-3.90 ¡ and between the methyl group C5M of V2:Van and
the ether bridge O2¤ of V1:Glu within the range of 3.40-3.59 ¡.
Hence, intermolecular sugar-sugar interactions may exert
nearly equal contribution to the stabilization of the dimer-
interface in these vancomycin complexes.
ring 6 of V1 and the other between the hydroxymethyl C6¤ of
glucose of V1 and ring 6 of V2. It should be noted here that
when larger ligands (NAAA and DALAA) bind to antibiotic,
the intra-dimer closest contact between C5M of V2 and C63
of V1 becomes shorter compared with that for small ligands
(OAc, AcG, DLac, AcDLac, phosphate, or AcDA): the
average distance of V2:Van C5M£V1:6 C63 contact is 3.57 ¡
[distributed from 3.50 to 3.72 ¡] for larger ligands, while the
corresponding value is 3.97 ¡ [with the range of 3.92-4.00 ¡]
for small ligands. On the other hand, the closest contact
between C6¤ of V1 and C63 or C65 of V2 becomes somewhat
longer when larger ligands bind: the V1:Glu C6¤£V2:6 C63 or
C65 average distance is 3.79 ¡ [with the range of 3.64-4.11 ¡]
for larger ligands while the corresponding average value is
3.60 ¡ [with the range of 3.49-3.68 ¡] for small ligands. As a
whole, when the high-affinity ligand binds, the stabilization of
the dimer-interface due to sugar-ring hydrophobic contacts
may increase to some extent.
(iv) Cross-linked aromatic side chain rings 4 and 6 within
each monomer are nearly perpendicular to each other (Figure 4
and Figure S-1). At the dimer-interface, there are two sets of
edge-to-face ³-interactions, one between the face of ring 4 of
V1 and the edge (C63) of ring 6 of V2 and the other between
the face of ring 4 of V2 and the edge (C63) of ring 6 of V1.
Table 2 shows that, when large ligands (NAAA, DALAA, or
even DALALac) bind to antibiotic, edge-to-face ³-interactions
become more effective (stronger) compared to those of small
ligands, especially prominent at the C-terminal side of V1 (see
V1:6 C63£V2:4 C41 distance). This may be, at least in part, due
to closer contacts between vancosamine of V2 and the face of
ring 6 of V1 for large ligands than for small ligands, as noted in
(iii), and vice versa.
(iii) There are two kinds of sugar-ring hydrophobic
interactions, one being intra-monomer and the other intra-
dimer (Figure 4 and Table S-2). The former may contribute to
stabilize structures of V1 or V2 monomers themselves, while
the latter may stabilize the structure of the dimer-interface.
Intra-monomer interactions in V1 occur between the methyl
substituent C3M of vancosamine and cross-linked aromatic side
chain of residue 6, and in V2 between vancosamine C3M and
the face of ring 2. Intra-dimer interactions occur at two sites,
one between the methyl group C5M of vancosamine of V2 and
In summary, when the high-affinity ligand binds, factors (i),
(iii), and (iv) work to make the dimer-interface tighten to some
extent.
Ligand-Binding Sites. As Figure 2 shows, each vanco-
mycin monomer in the phosphate and AcDA complexes forms

T. Kikuchi et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
395
Table 2. Selected Interatomic Distances (¡) around O66
Atoms at the Back-to-Back Dimer-Interface, Involving
Edge-to-Face Interactions between Cross-Linked Aromatic
Rings 4 and 6
hydrogen bonds, three of which are with amide nitrogens V2:1
N, V2:3 N, and V2:4 N and one with carbonyl oxygen V2:4 O
(Figure 2a for the phosphate complex and Figures 1 and 2b for
the AcDA complex). Table 3 lists hydrogen-bonding distances
between vancomycin and ligand molecules, along with those
of other vancomycin complexes whose crystal structures
have been determined. The phosphate ligand participates in a
weak P-O-H£O (V2:4) hydrogen-bonding in addition to three
conventional N-H£O (phosphate) hydrogen bonds. Moreover,
this phosphate oxygen atom makes close contact with the face
of aromatic ring 4 (see Figure 1 for the ring numbering) [the
shortest distance of 3.09 ¡ with C39], possibly through an
O-H£³ hydrogen-bonding (not shown in the figure), where the
hydrogen atom behaves as a bifurcated hydrogen-bonding
donor. These multiple interactions of the phosphate ligand with
the peptide backbone, especially, the formation of a P-O-H£O
(V2:4) hydrogen-bonding, may be responsible for higher
affinity of the phosphate ligand toward antibiotic over a
depsipeptide ligand N-acetyl-D-Ala-D-lactate that may suffer
from V2:4 O£O (ester oxygen of the ligand) electrostatic
repulsion when it binds to the peptide backbone of antibiotic.
In the AcDA complex, the AcDA ligand is well fitted into the
binding pocket by forming four conventional hydrogen bonds
with the antibiotic backbone to orient the D-Ala methyl group
so as to make close contacts with the front edge of ring 2
[3.85 ¡ to C20], the face of ring 4 [the perpendicular distance
of 3.78 ¡], and the chlorine atom of ring 6 [3.56 ¡] (See
Figure 6b for reference and Table S-3). These multiple hydro-
phobic interactions could be responsible for the higher binding-
affinity observed for AcDA, comparing with, for example, that
of N-acetylglycine [values of the binding-affinity obtained by
NMR measurements are 300 and 80 M¹1, respectively27].
Table 3 also shows that in crystals, there seems no apparent
correlation between the tightness due to the antibiotic-ligand
hydrogen-bonding and the molecular size.
Structure of Face-to-Face Arranged Self-Dimer. In the
crystal lattice, two V1 molecules form a face-to-face arranged
self-dimer V1-V1¤ with a crystallographic 2-fold axis that
passes through the water molecule located at the center of the
dimer-interface, as shown in Figure 5. V1 and V1¤ molecules
are connected to each other directly by a pair of hydrogen
bonds between the carbamoyl nitrogen of Asn residue 3 and the
carbonyl oxygen of residue 4 of its dimer partner across the
2-fold axis [V1:3 N¤2£V1¤:4 O = 2.84 ¡ for the phosphate
complex and 2.90 ¡ for the AcDA complex] and in addition,
indirectly by two pairs of water-mediated hydrogen-bondings
[V1:4 O£OW£V1¤:4 O; V1:7 O79£OW£V1¤:7 O79]. The side-
chain of the C-terminal Leu residue of V1 (or V1¤) molecule
locates at the hydrophobic pocket of V1¤ (or V1) molecule,
making multiple interactions involving a close contact with the
methyl substituent C5M of vancosamine of the dimer partner
[V1:1 C££V1¤:Van C5M = 4.14 and 4.25 ¡ for the phosphate
and AcDA complexes, respectively]. All of these intermolec-
ular interactions could contribute to the enhancement of the
formation as well as the stabilization of the face-to-face
arranged V1-V1¤ dimeric structure. A question arises here why
V1 but not V2 molecule forms the face-to-face arranged self-
dimer. A model-building study (using the the program ProFit25
by imposing the aglycone of V2 monomer on that of V1
V1:4 C41£ V1: O66£ V1:6 C63£
Ligand
Ref.
V2:6 C63
V2: O66
V2:4 C41
OAc
AcG
DLac
AcDLac
Phosphate
AcDA
Cu2+ ion
NAAAa)
DALAAb)
DALALacb)
4.06
4.17
4.06
4.04
4.03
4.05
3.84
3.93
3.93
3.92
4.60
4.72
4.57
4.61
4.32
4.64
3.92
4.25
4.23
4.34
4.54
4.59
4.50
4.49
4.31
4.43
3.99
4.12
4.09
4.19
13
15
15
15
this work
this work
16
6
6
6
a) Average values are given for 3 independent V1-V2 dimers.
b) Average values for 2 independent V1-V2 dimers.
an open bowl with the peptide backbone located on the bottom
(also shown in Figure S-2 for a V1 monomer and Figure S-3
for V2 monomers). The least-squares fits (performed using
the program ProFit25) of V1 and V2 monomers gave the
rms difference of the fitted atoms of 0.26 ¡ for the phosphate
complex and 0.24 ¡ for the AcDA complex, when the
heptapeptide-backbone atoms involving C¢ of each vancomy-
cin molecule are compared. This shows that the conformational
change on the ligand binding occurs to some extent, reflecting
in the width of the entrance of the binding pocket (estimated by
the distance between V:7 O79 and V:1 C£; see Figure 1 for the
atom numbering): 8.20 ¡ for V1 and 8.67 ¡ for V2 in the
phosphate complex, and the corresponding distances in the
AcDA complex are 8.15 and 8.55 ¡. On the other hand, the
least-squares fits of V1 in the phosphate complex and V1 in the
AcDA complex, and V2 in the phosphate complex and V2 in
the AcDA complex are 0.06 and 0.14 ¡, respectively, showing
no conformational difference between two V1 monomers or
minor difference between two V2 monomers. It is of interest
to note here that the width of the entrance of the ligand-
binding pocket decreases when the ligand becomes larger:
OAc13 [8.78 ¡] ¼ AcDA [8.55 ¡] ¼ NAAA6 [7.88 to 8.34 ¡
(average, 7.99 ¡)] ¼ DALAA6 [7.06 to 8.05 ¡ (average,
7.59 ¡)].
In the structures of V1 monomers in both the phosphate and
AcDA complexes, no ligand locates at the binding pocket.
Instead, the carbamoyl side-chain group of Asn residue
participates in the formation of two intramolecular N-H£O
hydrogen bonds with two amide nitrogens V1:3 N and
V1:4 N of the peptide backbone: V1:3 N£V1:3 O¤1 and
V1:4 N£V1:3 O¤1 distances are 2.81 and 2.80 ¡, respectively,
for the phosphate complex and 2.82 and 2.81 ¡ for the AcDA
complex. Loll et al. suggested13 that the Asn residue, which is
essential for antimicrobial activity,26 acts as an intramolecular
surrogate ligand in the absence of the ligand, thereby promot-
ing the ligand-binding activity by preventing solvation or
facilitating desolvation of the binding site.
In the structures of V2 monomers, a phosphate ligand or an
AcDA ligand binds to the peptide backbone through four

396
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
Structures of Two Vancomycin Complexes
Table 3. Hydrogen-Bonding Distances (¡) between Vancomycina) (V) and Ligand (L) Molecules in Crystal Structures of
Vancomycin Complexes
Vancomycin
Ligand
OAcb) AcGb) DLacb)
AcDLacb)
Phosphateb)
AcDAb) NAAAc) DALAAd)
V1:2 N
V2:2 N
V1:3 N
V2:3 N
V1:4 N
V2:4 N
V1:4 O
V2:4 O
V1:7 N
V2:7 N
L: OXT
L: OXT
L: O
L: O
L: O
L: O
L: N
L: N(Oe),f)
L: O
2.79
2.85
2.80
2.92
2.86
2.83
2.84
2.89
2.88g)
2.87
2.79
2.76
2.99
2.96
2.81
2.77
2.98
2.90
2.88
2.88
2.81
2.88
2.91
2.79
3.01
2.98
2.94
2.81
2.90
2.93
2.77e)
2.82
2.92
2.93
2.98
2.83
2.90
3.15f)
2.77
2.88
2.86
2.90
)
L: O
a) Vancomycin takes two types of monomers in which the vancosamine sugar lies over the ligand-binding pocket in the V1
monomer, while the glucose occupies the corresponding position in the V2 monomer (see the text). b) No ligand is bound to
V1. c) The NAAA complex involves 6 independent V-L pairs. Average values are given for 3 V1-L and 3 V2-L pairs. d) The
DALAA complex involves 6 independent V-L pairs. Average values are given for 4 V1-L and 2 V2-L pairs. e) Hydroxy
oxygen of DLac ligand. f) Protonated oxygen of phosphate ligand. g) Average value is given for 2 V1-L pairs.
the triclinic form, while it is 2:1 in the present tetragonal form.
The most interesting structural feature in the triclinic form is
the first observation that vancomycin forms the ligand-
mediated face-to-face arranged dimer but in this case, two
incorporated ligands do not come into any direct contact. A
modified ligand-mediated face-to-face dimeric structural motif
has subsequently been observed in complexes with longer
ligands (high-affinity ligands), such as a balhimycin complex5
with L-Lys-D-Ala-D-Ala, or a degluco-balhimycin complex5
with L-Ala-D-Glu-£-L-Lys-D-Ala-D-Ala and vancomycin
complexes with NAAA6,7 and DALAA,6 where the two
incorporated ligands in the binding pockets are further self-
associated by forming N-H£O hydrogen bonds between
peptide backbones. This ligand-mediated face-to-face oligo-
merization is of special interest in that, as Scheldrick et al. have
argued,5 such a structural motif may provide a realistic model
Figure 5. Stereoview showing the face-to-face arranged
self-dimer V1-V1¤ in the AcDA complex. The dimeric
structure is stabilized by direct or water-mediated hydrogen
bonds between V1 and V1¤ monomers, shown in red or
gray dotted lines, respectively, across a crystallographic
dyad axis passing through the water molecule (pink
sphere), the former being a pair of V1:3 N¤2£V1¤:4 O
hydrogen bonds and the latter being two pairs of
V1:4 O£OW£V1¤:4 O and V1:7 O79£OW£V1¤:7 O79 hy-
drogen bonds. Note that the Leu side-chain occupies the
ligand-binding site of its dimer partner.
for the prevention of cell-wall crosslinking by antibiotic
binding. Detailed comparison between the two forms is not
possible since neither PDB nor CCDC data are available for the
triclinic form, and thus in this study, for referring to the data
of the AcDA complex, we exclusively deal with the present
tetragonal form.
Non-Equivalence in the Ligand-Binding Affinities at the
Two Binding Sites in the Back-to-Back Arranged Dimer.
We now try to address a question for the absence of ligand at
the binding pocket in V1 monomer in the present and other
low-affinity ligand complexes, by examining the ligand-bind-
ing affinity at the two binding sites in V1- and V2-type
monomers, where as noted above, vancosamine locates over
the ligand-binding pocket in the V1 monomer while glucose
occupies the corresponding position in the V2 monomer. A
comparison of the tightness of the antibiotic-ligand (L)
interface at the two binding sites in V1- and V2 monomers
in the NAAA6 and DALAA6 complexes, where both sites are
ligand-bound, shows no significant difference between the
corresponding hydrogen-bonding distances at the two binding
sites (Table 3). The only exception is for the V:3 N£L:O
hydrogen bond in the NAAA complex in which the V:3 N£
L:O distance [2.80 ¡] in V1 monomer is shorter than that
monomer) shows that when V2 molecules form the face-to-face
arranged self-dimer V2-V2¤, the glucose sugar locates at a
remote position from the Leu side chain of its dimer partner (as
shown in Figure S-4), thereby making hydrophobic interaction
less effective [the nearest interatomic V2:1 C££V2¤:Glu C5¤
distance of 5.71 ¡ for the AcDA complex].
Comparison of the Two AcDA Complexes with Tetrag-
onal and Triclinic Crystal Forms.
The triclinic AcDA
complex12 involves four independent vancomycin monomers
(two independent back-to-back dimers), each of which incor-
potates a ligand. Thus, the ratio of vancomycin:ligand is 1:1 in

T. Kikuchi et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
397
Possible Scenarios for the Complex Formation. In the
low-affinity ligand complexes, a model-building study (using
the program Profit25) shows that sugar-ligand steric constraint
is negligible when the ligand binds to V1 monomer [for
example, in the phosphate complex, the calculated nearest
V1:Van C5M£O (phosphate ligand) contact is 4.45 ¡]. Accord-
ingly, the absence of the ligand in V1 is most probably due to
an inherent property of vancomycin to form a face-to-face
arranged V1-V1¤ self-dimer, where each ligand-binding site is
occupied by the side-chain of the Leu residue of its dimer
partner, thereby preventing the ligand from binding to V1
(Figure 5). On the other hand, the absence of the ligand in V1
in the present AcDA complex is somewhat surprising since
both sites are occupied in the triclinic vancomycin-AcDA
complex.12 The formation of these two different crystal forms
may be interpretable by considering the crystallization pro-
cesses associated with the crystallization conditions. With the
triclinic AcDA complex (crystallized from 27 mM antibiotic
and 0.1 M AcDA12), we assume that a back-to-back dimer may
form at first, followed by the ligand binding to V2 due to the
lower ligand-binding affinity toward V1 than V2. As a next
step, the ligand binding to V1 (of the V1-V2 dimer where the
ligand-binding site of V2 is occupied) and the V1-V1¤ self-
dimerization (of two V1-V2 dimers with V2 being ligand-
bound) may compete with each other, and as a result, since
concentrations are higher for free-ligand than V1-V2 dimer-
species (with V2 being ligand-bound) by at least twice, the
ligand binding to V1 overcomes the V1-V1¤ self-dimerization
to yield a vancomycin complex with both sites occupied. On
the other hand, with the present AcDA complex (crystallized
from equimolar (50 mM) antibiotic and N-acetyl-D-Ala-D-
lactate), the hydrolysis of N-acetyl-D-Ala-D-lactate (to give
AcDA) takes some time during which the V1-V1¤ self-dimer
forms at first, and once this is formed, due to the sugar-ligand
repulsive close contact noted above, the ligand-binding affinity
of AcDA toward V1 is not enough to displace the side-chain of
the Leu residue locating at the ligand-binding site.
(a)
(b)
Figure 6. Multiple hydrophobic contacts between D-Ala
methyl group of the ligand and the antibiotic molecule for
V1 monomer (a) and V2 monomer (b) in the NAAA
complex,6 drawn with dotted lines.
[2.92 ¡] in V2 monomer, whereas the corresponding distance
is comparable to each other in V1 and V2 monomers of the
DALAA complex. Thus the absence of the ligand in V1
monomer in these low-affinty ligand vancomycin complexes
is not explicable in the term of the peptide-backbone-ligand
affinity, which can be estimated to be essentially equivalent
between the two sites.
On the other hand, for high-affinity ligands, the formation of
a back-to-back arranged V1-V2 dimer and the ligand binding
to both sites may cooperatively proceed, and V1-V2 dimers
subsequently self-associate by forming hydrogen bonds be-
tween the two antiparallel running ligands to give a ligand-
mediated face-to-face arranged oligomer, as observed in the
NAAA6,7 and DALAA6 complexes.
On the other hand, the sugar-ligand interaction seems to be
less favorable for V1 monomer than for V2 monomer, since, in
the NAAA and DALAA complexes, very short close contact
[distributed from 3.42 to 3.82 ¡] is observed in V1 monomer
between the methyl side-chain group of the terminal D-Ala
residue of the ligand and the methyl substituent (C5M) of
vancosamine (Figure 6a and Table S-3). This indicates that the
methyl-methyl close contact may suffer from considerable
steric hindrance, judged from the radius of the methyl group of
2.0 ¡.28 On the other hand, in V2 monomer, multiple hydro-
phobic contacts occur between the D-Ala methyl group of
the ligand and the edge of glucose (ring-atoms C1¤ or O5¤)
(Figure 6b) [interatomic distances in the normal range of
4.03-4.59 ¡ (Table S-3)]. This may contribute to the stabiliza-
tion of the antibiotic-ligand interface.
Conclusion
Despite many trials under a number of crystallization
conditions, co-crystallization of vancomycin and N-acetyl-D-
Ala-D-lactate (as a cell-wall precursor analog) has failed. We
propose that, since N,N¤-diacetyl-L-Lys-D-Ala-D-lactate does
bind to vancomycin,6 the third Lys residue from the C-terminus
of the ligand is necessary as a minimal set for the complex
formation with the vancomycin-resistent precursors terminating
in -D-Ala-D-lactate, though we are aware that more direct and
quantitative data on the affinity of vancomycin toward both
N-acetyl-D-Ala-D-lactate and N,N¤-diacetyl-L-Lys-D-Ala-D-
lactate are indispensable to confirm our hypothesis.
Both the present phosphate and AcDA complexes belong to
a family of low-affinity ligand complexes, where two vanco-

398
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
Structures of Two Vancomycin Complexes
1211, 1174, 1097, 1072, 752, 698; 1H NMR (270 MHz, CDCl3): ¤
7.41-7.24 (m, 10H), 5.38-4.99 (m, 5H), 5.27 (br d, J = 7.3 Hz,
1H), 4.43 (dq, J = 7.3, 7.3 Hz, 1H), 1.52 (d, J = 7.1 Hz, 3H), 1.41
(d, J = 7.3 Hz, 3H); 13C NMR (67.8 MHz, CDCl3): ¤ 172.5, 170.1,
155.6, 136.2, 135.1, 128.6, 128.5, 128.2, 128.14, 128.05(2), 69.2,
67.1, 66.9, 49.3, 18.4, 16.7; Anal. Calcd for C21H23NO6: C, 65.44;
H, 6.02; N, 3.63%. Found: C, 65.21; H, 6.12; N, 3.82%.
mycin molecules form a back-to-back arranged dimer V1-V2
with only one of the two ligand-binding sites occupied by a
ligand in V2. In this study, we have found that (i) when the
high-affinity ligand binds, three structural factors (hydrogen-
bonding interactions between the two peptide-backbones,
hydrophobic interactions between the vancosamine sugar and
the cross-linked ring 6, and hydrophobic edge-to-face ring-ring
interactions between cross-linked rings 4 and 6) could enhance
the stabilization of the back-to-back dimer-interface to some
extent, and (ii) the ligand-binding affinity between the two
binding sites in the back-to-back arranged dimer may be less
for V1 monomer than V2 monomer due to the sugar
(V1:Van C5M)£ligand (Me group of D-Ala) steric constraint.
The absence of the ligand binding to V1 in the low-affinity
ligand complexes is most likely due to the formation of the
face-to-face arranged self-dimer V1-V1¤, where the side-chain
of the Leu residue of V1 (V1¤) locates at the ligand-binding
pocket of V1¤ (V1) as a surrogate ligand. The suger-ligand
steric constraint may be an additional reason for the absence of
the ligand in V1 of the present AcDA complex. In this context,
solution studies on whether the formation of the face-to-face
arranged self-dimer V1-V1¤ is an inherent nature of vancomy-
cin for low-affinity ligands and especially when ligand is free,
and whether there exists any difference in the ligand-binding
affinity between the two binding sites in the back-to-back
arranged dimer when AcDA binds, are of special interest and
remain to be undertaken.
Synthesis of N-Acetyl-D-Ala-D-Lactate: To a solution of
benzyl N-Z-D-Ala-D-lactate (4.00 g, 10.4 mmol) in MeOH
(25 mL) was added a suspension of 10% Pd-C (1.42 g) in EtOAc
(14 mL). The reaction mixture was stirred vigorously for 1.5 h
under H2 atmosphere at rt. The mixture was filtered through a pad
of Celite, and the filter cake was eluted with MeOH and distilled
water. The filtrate was concentrated in vacuo. To an ice-cooled
solution of the residue was added successively NaHCO3 (1.36 g,
16.2 mmol) and acetic anhydride (1.50 mL, 15.9 mmol), and the
reaction mixture was stirred for ca. 20 h at rt. To the mixture was
added 3 M HCl (ca. 5 mL) at 0 °C, and the resulting mixture was
extracted several times with EtOAc. The extract was dried
(MgSO4), and concentrated in vacuo. The residue was purified
by recrystallization from EtOAc-hexane to give N-acetyl-D-Ala-
24:8
D-lactate (1.25 g, 59%) as colorless rods; mp 147-149 °C; ½¡ꢀD
¹1
+124° (c 0.730, H2O); ¯max (KBr)/cm 3400, 3001, 2534, 2501,
1751, 1728, 1622, 1541, 1448, 1377, 1236, 1213, 1167, 1095,
1038, 633, 563, 484, 413; 1H NMR (270 MHz, D2O, referred to the
residual signal of HOD at 4.65 ppm): ¤ 4.98 (q, J = 7.1 Hz, 1H),
4.30 (q, J = 7.3 Hz, 1H), 1.89 (s, 3H), 1.40 (d, J = 7.1 Hz, 3H),
1.32 (d, J = 7.5 Hz, 3H); 13C NMR (67.8 MHz, D2O, acetone as
internal reference at 30.6 ppm): ¤ 175.3, 174.6, 174.5, 70.7, 48.9,
21.8, 16.4, 16.2; Anal. Calcd for C8H13NO5: C, 47.29; H, 6.45; N,
6.89%. Found: C, 47.31; H, 6.49; N, 7.00%.
Experimental
Synthesis of N-Acetyl-D-Ala-D-Lactate. N-Acetyl-D-Ala-
D-lactate was prepared according to the literature.29 General
information: Mp is uncorrected. NMR spectra were recorded on a
JEOL GSX-270 spectrometer. 1H NMR and 13C NMR chemical
shifts are reported in ¤-values based on internal tetramethylsilane
(¤H = 0), or solvent signal (CDCl3 ¤C = 77.0) as reference unless
otherwise indicated. IR spectra were recorded on a HORIBA
FT-720 Fourier-transform infrared spectrometer. Flash silica gel
column chromatography was carried out on Merk Kieselgel 60
(230-400 mesh), Art. Nr. 9385. Optical rotations were measured
Crystallization and Data Collection.
Vancomycin was
purchased from Wako Junyaku and used without further purifica-
tion. Crystals were grown by hanging drop vapor diffusion at room
temperature. A total of thirty different reservoirs were tested in
co-crystallization experiments of vancomycin and N-acetyl-D-
Ala-D-lactate using Crystal Screen kits,31 yielding two crystalline
compounds, one being a phosphate complex and the other being an
AcDA complex.
Crystallization of the Phosphate Complex under the Pres-
ence of N-Acetyl-D-Ala-D-Lactate at pH 6.5: The drop solution
contained 2 ¯L of 50 mM vancomycin, 2 ¯L of 50 mM N-acetyl-D-
Ala-D-lactate, and 2 ¯L of reservoir solution. The 500 ¯L reservoir
solution contained 2.0 M NaCl, 0.2 M Na/K phosphate, and
0.10 M 2-(morpholino)ethanesulfonic acid (pH 6.5). Bipyramidal
crystals grew to about 0.6 © 0.6 © 0.8 mm3 in a few weeks.
Crystallization of the AcDA Complex, Derived from the
Hydrolysis of N-Acetyl-D-Ala-D-Lactate at pH 4.5: The drop
solution contained 2 ¯L of 50 mM vancomycin, 2 ¯L of 50 mM N-
acetyl-D-Ala-D-lactate, and 2 ¯L of reservoir solution. The 500 ¯L
reservoir solution contained 2.0 M NaCl, and 0.1 M sodium acetate
(pH 4.5). Bipyramidal crystals grew to about 0.2 © 0.3 © 0.6 mm3
in a few weeks.
Data Collection: One crystal was used for the data collection
for each of the phosphate and AcDA complexes. The crystal was
flash-cooled by a stream of cold nitrogen gas at 95 K with the
addition of glycerol as cryoprotectant. Diffraction data were
measured up to 1.10 ¡ for the phosphate complex and 0.95 ¡ for
the AcDA complex at beam line BL38B1 at the Japan Synchrotron
Radiation Research Institute (Spring-8, Hyogo). Oscillation
angle and exposure time were 1.0°/frame and 15 s/frame,
respectively. Integrated intensities were calculated using the
on a Rudolph Research Analytical AUTOPOL V polarimeter, and
¹1
[¡]D-values are given in units of 10¹1 deg cm2 g
.
Synthesis of Benzyl N-Benzyloxycarbonyl-D-Alanyl-D-
Lactate (Benzyl N-Z-D-Ala-D-Lactate): To an ice-cooled
solution of N-Z-D-Ala (3.80 g, 17.0 mmol), benzyl D-lactate
(3.07 g, 17.0 mmol; prepared by the iodine-catalyzed transester-
ification30 from methyl D-lactate and benzyl alcohol) and N,N-
dimethylaminopyridine (DMAP) (2.09 g, 17.1 mmol) in dry
CH2Cl2 (100 mL) was added dropwise a solution of dicyclohexyl-
carbodiimide (DCC) (3.89 g, 18.9 mmol) in dry CH2Cl2 (20 mL),
and the reaction mixture was stirred for ca. 22 h at rt. During this
time, an additional amount of N-Z-D-Ala (194 mg, 0.867 mmol)
and N,N-dimethylaminopyridine (DMAP) (108 mg, 0.887 mmol)
were supplied. The mixture was filtered through a pad of Celite.
The filter cake was eluted with EtOAc. The filtrate was washed
successively with saturated NaHCO3 aq., 0.5 M HCl, saturated
NaHCO3 aq. and brine, and dried (MgSO4), and concentrated in
vacuo. The residue was purified by flash column chromatography
(hexane:EtOAc = 11:1) to give N-Z-D-Ala-D-lactic acid benzyl
26:4
ester (6.32 g; 96% yield) as a colorless oil; ½¡ꢀD +36.7° (c 1.15,
¹1
CHCl3); ¯max (neat)/cm 3346, 1749, 1724, 1525, 1456, 1252,

T. Kikuchi et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 4 (2010)
399
Table 4. Statistics of Crystal, Data Collection and
Refinement
phosphate ion, three chloride ions, a sodium ion, 18 fully-occupied
water molecules, and a half-occupied water molecule in the
phosphate complex, while two vancomycin monomers, an AcDA
ligand, three chloride ions, a sodium ion, 28 fully-occupied water
molecules, and 2 half-occupied water molecules in the AcDA
complex. In the phosphate complex, the phosphate ion exists as
Ligand
Phosphate
AcDA
Contents of an asymmetric unit cell
Vancomycin
(FW)
Ligand
(FW)
Ion
2 © C66H77N9O24Cl2 2 © C66H77N9O24Cl2
2¹
HPO4 as evidenced by its molecular dimensions, in accordance
(2 © 1451.4)
[HPO4]2¹
(96.0)
(2 © 1451.4)
[C5H8NO3]
with the crystallization conditions (pH 6.5). Crystallographic
data have been deposited with Cambridge Crystallographic Data
Centre: Deposition numbers CCDC-728179 and -728180 for the
phosphate and AcDA complexes, respectively. Copies of the data
can be obtained free of charge via http://www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the Crystallographic Data Centre,
12, Union Road, Cambridge, CB2 1EZ, U.K.; Fax: +44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk).
¹
(130.1)
¹
¹
3 © Cl , Na+
3 © Cl , Na+
(FW)
Water molecule
(129.3)
23 (full) and
1 (partial)
95
(129.3)
32 (full) and
2 (partial)
95
Temperature/K
Wavelength/¡
0.700
0.700
Space group
Unit cell parameters
P43212
P43212
Supporting Information
Lists of distances between disaccharides (Table S-1) and sugar-
ring distances (Table S-2), at the back-to-back dimer-interface. A
list of contacts between the methyl group of the D-Ala moiety of
the ligand and the antibiotic at the ligand-binding site (Table S-3).
Figures showing a V1 monomer (Figure S-1), V2-monomers
(Figure S-2), and edge-to-face arranged rings 4 and 6 at the back-
to-back dimer-interface (Figure S-3). A figure showing a model
for a face-to-face arranged self-dimer V2-V2¤ (Figure S-4). These
materials are available free of charge on the web at http://www.
csj.jp/journals/bcsj/.
a/¡
b/¡
28.49(3)
28.49(3)
65.42(7)
53093(91)
8
2.14
42.4
14.8-1.10
78896
28.22(3)
28.22(3)
65.69(7)
52299(90)
8
2.10
41.5
28.2-0.95
157178
17050
97.4
c/¡
V/¡3
Z
Vm
Solvent content/%
Resolution range/¡
Observed reflections
Independent reflections 11585
Completeness/%
Multiplicity
99.8
6.8
0.069
9.98-1.10
9.2
0.063
9.98-0.95
References
Rmerge (I)
1
D. H. Williams, B. Bardsley, Angew. Chem., Int. Ed. 1999,
Resolution range/¡
Reflections used for
refinement
Cut off (·)
Completeness/%
Final R
38, 1172, and references therein.
10896
15885
2
2291.
3
P. Courvalin, Antimicrob. Agents Chemother. 1990, 34,
4
4
H. R. Perkins, Biochem. J. 1969, 111, 195.
94.2
0.128
90.9
0.127
4
D. H. Williams, M. P. Williams, D. W. Butcher, S. J.
Hammond, J. Am. Chem. Soc. 1983, 105, 1332.
C. Lehmann, G. Bunkóczi, L. Vértesy, G. M. Sheldrick,
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5
program MOSFLM,32 and scaling was performed using the
program SCALA33 of the CCP4 program package. Crystal data
and data collection statistics, together with refinement statistics, are
listed in Table 4.
6
7
Structure Solution and Refinement.
Initial models were
obtained by molecular replacement (MR) with the program
AMORE34 of the CCP4 program package. As a search model,
the vancomycin dimer of the PDB code 1C0R35 was used. MR
calculations with the standard protocol gave the solution with R
values of 33.6% for the phosphate complex and 32.1% for the
AcDA complex. The crystallographic refinements were performed
with the program REFMAC536 of the CCP4 program package. The
model was refined under chemical restrictions with a restriction file
that was generated referring the library file of vancomycin,
VAN.cif, of the CCP4 program suite. The electron density maps,
2mFo ¹ dFc map, Fo ¹ Fc map, and omit map were calculated and
checked by the program XTALVIEW,37 revealing a phosphate ion
in the phosphate complex or an AcDA ligand in the AcDA
complex, in addition to three chloride ions, a sodium ion, and
water molecules in both complexes. After a few cycles of
refinement with isotropic thermal parameters, the anisotropic
refinement gave the final R values of 12.8% for the phosphate
complex and 12.7% for the AcDA complex. The crystallograph-
ically asymmetric unit contains two vancomycin monomers, a
8
9
C. T. Walsh, Science 1993, 261, 308.
N. E. Allen, D. L. LeTourneau, J. N. Hobbs, Jr., J. Antibiot.
1997, 50, 677.
10 T. I. Nicas, C. T. Cole, D. A. Preston, A. A. Schabel, R.
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