From the Department of Basic and Applied Molecular
Biotechnology, Division of Food and Biological Science and the
§ Laboratory of Quality Design of Exploitation, Division of
Agronomy and Horticultural Science, Graduate School of Agriculture,
Kyoto University, Uji, Kyoto 611-0011, Japan
Received for publication, September 27, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Sphingomonas sp. A1 possesses
a high molecular weight (HMW) alginate uptake system composed of a
novel pit formed on the cell surface and a pit-dependent
ATP-binding cassette (ABC) transporter in the inner membrane.
The transportation of HMW alginate from the pit to the ABC transporter
is mediated by the periplasmic HMW alginate-binding proteins AlgQ1 and
AlgQ2. We determined the crystal structure of AlgQ2 complexed with an
alginate tetrasaccharide using an alginate-free (apo) form as a search
model and refined it at 1.6-Å resolution. One tetrasaccharide was
found between the N and C-terminal domains, which are connected by
three extended hinge loops. The tetrasaccharide complex took on a
closed domain form, in contrast to the open domain form of the apo
form. The tetrasaccharide was bound in the cleft between the domains
through van der Waals interactions and the formation of hydrogen bonds. Among the four sugar residues, the nonreducing end residue was located
at the bottom of the cleft and exhibited the largest number of
interactions with the surrounding amino acid residues, suggesting that
AlgQ2 mainly recognizes and binds to the nonreducing part of a HMW
alginate and delivers the polymer to the ABC transporter through
conformational changes (open and closed forms) of the two domains.
ABC1 transporters
are widely found in bacteria to
humans. All of them consist of two membrane proteins spanning the
cytoplasmic membrane and forming a translation pore with two ATPase
molecules providing the energy for accumulation of a substrate inside
the cell membrane. Furthermore, ABC importers of Gram-negative bacteria depend on a periplasmic substrate-specific binding protein.
A Gram-negative soil bacterium, Sphingomonas sp. A1, was
isolated as a potent producer of an alginate lyase, which catalyzes the
depolymerization of a high molecular weight (HMW) alginate (average
molecular size, 25,700 Da) (1). Sphingomonas sp. A1 cells
are covered with many large plaits (2). When they are assimilating an
alginate, pits (0.02-0.1 µm in diameter) are formed on their surface
through reconstitution and/or rearrangement of these plaits, and the
biopolymer is concentrated in the pits (3). The alginate thus
accumulated is then delivered through the action of periplasmic
alginate-binding proteins (AlgQ1 and AlgQ2) to a
pit-dependent ABC transporter. An alginate-specific ABC
transporter consists of AlgM1 and AlgM2 as membrane-spanning permeases
and AlgS as an ATP-binding protein (4).
An alginate is a polymer comprising The HMW alginate-binding proteins of Sphingomonas sp. A1,
AlgQ1 and AlgQ2, are members of a large group of periplasmic binding proteins of Gram-negative bacteria (4). AlgQ1 and AlgQ2 exhibit high
amino acid sequence similarity to each other (74% homology), although
their sequence identity with other binding proteins is less than 30%.
Both AlgQ1 and AlgQ2 function in a monomeric form of 57 kDa. Although
binding proteins have a diverse set of ligands (e.g.
monosaccharides, oligosaccharides, oxyanions, amino acids, oligopeptides, and vitamins), an extraordinary feature of AlgQ1 and
AlgQ2 is that they bind to a macromolecule with similar high affinities, the Kd values being around
10 Periplasmic binding proteins of ABC transporters are found in various
kinds of prokaryotic microbes (8), and the crystal structures of some
of these proteins have been determined. Although they exhibit little
sequence similarity and are different sizes, ranging from 20 to 60 kDa
(8), they have similar overall structures composed of two globular
domains with a deep cleft between the domains. The binding of
substrates periplasmic proteins favors their closure via large scale
hinge bending motions (8). These movements are required for productive
interaction with the membrane permeases. The structures of periplasmic
binding proteins of Gram-negative bacteria have many common features as
described above, and almost all of them so far analyzed were found to
be responsible for the binding of small solutes such as maltose,
ribose, amino acids, peptides, and metals (8). However, AlgQ1 and AlgQ2
can bind a macromolecule, and this HMW alginate-specific ABC
transporter has two binding proteins (AlgQ1 and AlgQ2), although
bacterial ABC transporters generally have one periplasmic binding
protein. Because alginate is composed of two sugar monomers (M and G), AlgQ1 and AlgQ2 may have specificity for either sugar or a certain polysaccharide arrangement. Therefore, the structural analysis of a
periplasmic binding protein having affinity with macromolecules might
provide a new insight into the molecular mechanism underlying macromolecule transport through the action of periplasmic binding protein-dependent ABC transporters.
To elucidate the structural and functional relationship of periplasmic
binding proteins in HMW alginate transport, we have determined the
x-ray crystal structure of AlgQ2 complexed with an alginate
tetrasaccharide and revealed the structure of the alginate binding site
as well as the mode of alginate binding accompanying a large
conformational change.
Purification, Crystallization, and X-ray Diffraction--
The
methods used for the purification of AlgQ2 have been described
previously (9). Briefly, AlgQ2 was purified from Escherichia coli cells transformed with the algQ2 gene by cation
exchange column chromatography, dialyzed against 20 mM
sodium HEPES buffer, pH 7.0, and then concentrated to ~15 mg/ml. An
alginate tetrasaccharide was prepared through depolymerization of HMW
alginate with alginate lyase A1-III (10). Cocrystals of AlgQ2 bound
with the alginate tetrasaccharide were obtained by the hanging drop
vapor diffusion method. The solution for a cocrystallization drop was
prepared at 20 °C on a siliconized coverslip by mixing 3 µl of the
protein solution (15 mg/ml) with an equal volume of mother liquor
comprising 30% polyethylene glycol 4000, 0.2 M ammonium
acetate, 1 mM alginate tetrasaccharide, and 0.1 M sodium citrate buffer, pH 5.6. Diffraction data for a
crystal of AlgQ2 complexed with the tetrasaccharide were collected up
to 1.6 Å with Structure Determination and Refinement--
The crystal
structure of AlgQ2 complexed with the tetrasaccharide (holo-AlgQ2) was
solved by the molecular replacement method using program CNS (13). The
coordinates of the ligand-free form of AlgQ2 (9) (apo-AlgQ2; RCSB
Protein Data Bank (14), under accession number 1KWH) were used as a
search model. Model building was performed with program TURBO-FRODO
(AFMB-CNRS, France) on a Silicon Graphics Octane computer. Simulated
annealing refinement was carried out with this model using 61.6-2.5-Å
resolution data with program CNS (13). Fo
The stereo quality of the model was assessed using program PROCHECK
(15). Ribbon plots were prepared using programs MOLSCRIPT (16),
RASTER3D (17), and GRASP (18). These molecular models were superimposed
using the fitting program implemented in TURBO- FRODO.
Structure Determination--
A crystal of holo-AlgQ2 (0.1 mm × 0.1 mm × 0.05 mm) was obtained in 2 weeks by the hanging drop
vapor diffusion method. The space group of the crystal was determined
to be P21, with unit cell dimensions of
a = 95.63 Å, b = 53.88 Å,
c = 114.92 Å, and Overall Structure of AlgQ2 Complexed with an Alginate
Tetrasaccharide--
Fig. 1a
shows a C
The overall structure of holo-AlgQ2 was similar to that of apo-AlgQ2,
except for the disposition of the N and C domains (N domain, residues
1-133 and residues 310-400; C domain, residues 134-309 and residues
401-492). The designation of secondary structure elements of
holo-AlgQ2 is the same as that for apo-AlgQ2 (for a description, see
Ref. 9). Briefly, holo-AlgQ2 is composed of two globular domains (N and
C domains) that form an
Although almost all binding proteins of ABC transporters consist of two
domains, and a deep cleft corresponding to a substrate binding site is
formed between the two domains, differences in the transitions or
crossovers from one domain to the other are observed in the folding
topology of the binding protein structures. Based on these differences,
the binding proteins are divided into two types (groups I
and II) (21). In group I, the transitions from domain to domain are
strand to helix for the first two crossovers and strand to strand for
the third crossover e.g. galactose-binding protein (GBP) and
L-arabinose-binding protein (ABP), and in group II, the
first two are strand to strand and the third is helix to helix,
e.g. maltose-binding protein (MBP) and sulfate-binding protein (21). Therefore, AlgQ2, having a sheet topology and crossover
connections, is classified into group II.
In contrast to apo-AlgQ2, whose N and C domains are wide open, the
complex form with the tetrasaccharide was found to be a closed one as
reported for other substrate-binding proteins of ABC transporters. For
example, in MBP, hinge bending between the two domains involves
rotation of ~35° (22). Between the apo and holo forms, the root
mean square deviation of equivalent C Alginate Binding Site--
The binding site for alginate in AlgQ2
is located in the cleft between the two globular domains (N and C
domains) (Fig. 1a). AlgQ2 has an especially deep cleft
compared with other periplasmic binding proteins. In the open form of
AlgQ2, basic residues, mostly Arg, are on the surface around the cleft
in each domain, and aromatic residues are located deep in the cleft
(Fig. 2). When AlgQ2 is in the closed
form, basic residues face the center of the cleft. The positively
charged residues in the cleft might facilitate the attachment of the
negatively charged alginate molecule in the cleft and hold the
substrate in a proper position for binding. These changes show that
basic residues, especially Arg, play a role as sensors for alginate and
facilitate the efficient binding of alginate.
The bound alginate residues (tetrasaccharide) were identified as
In both molecules A and B, M4 is an
The bound tetrasaccharide molecule undergoes interactions with the
surrounding amino acid residues (Fig. 4).
The hydrogen bond interactions between the bound tetrasaccharide and
AlgQ2 are listed in Table III. Consistent
with the involvement of many charged residues in sugar binding sites,
basic and acidic side chains provide many of the nitrogen and oxygen
atoms capable of hydrogen bond formation, respectively. Side chains
with polar planar groups (Asn, Asp, Glu, Arg, and His) are the ones
most often used for hydrogen bonding to carbohydrates (26). The number of hydrogen bonds associated with the tetrasaccharide is 31 (
Comparison of the structures showed that two water molecules in the
apo-protein are replaced on carbohydrate binding. These two water
molecules, WAT584 and WAT617, occupy positions O61 and O62 of
All residues binding with
The C-C contacts between AlgQ2 and the tetrasaccharide are listed in
Table IV. The number of C-C contacts is
39 (
To analyze the specificity at the S1 subsite, saturated or unsaturated
glucuronate or saturated mannuronate was placed at the Hinge Bending Motion of AlgQ2--
The rotations around main chain
dihedrals in the three interdomain connections are related to the
opening and closing of AlgQ2. Movements in the three hinge segments are
greatest at residues 133-136 (L-SA1:SC3), 294-314 (L-SC4:SA3), and
399-401 (L-H19:H20) (L, loop; SA,
The hinge region of AlgQ2 is shown in Fig.
5. The hydrogen bond between
Glu396 (which is an element of H19 in the N domain) and
Arg309 (which is an element of L-SC4:SA3) is maintained in
both the opened and closed forms and thus stabilizes the interaction
between H19 and L-SC4:SA3 (Fig. 5 and Table
V). In the closed form, H19 and L-SC4:SA3
are close to the tetrasaccharide in the cleft, and the hydrogen bond
formed between Glu396-OE2 and
Therefore, the hinge motion of AlgQ2 can be predicted to be as follows.
When Glu396 forms a hydrogen bond with In periplasmic binding protein-dependent
transport systems, a soluble binding protein is the first component to
interact with the substrate to be transported, acting as a high
affinity receptor for the substrate in the periplasm. A little
understanding of how binding proteins function in transport has been
obtained through studies on the MBP system (22, 26-28). In this
system, MBP undergoes a ligand-induced conformational change that has
been observed in both the presence and absence of a ligand, and MBP
becomes tightly bound to the membrane transporter.
The way in which a tetrasaccharide and HMW alginate bind to AlgQ2 is of
interest. Because an alginate is a highly polar organic molecule,
hydrogen bonding is the dominant interaction in protein-carbohydrate complex formation. Thus, on complex formation, the solvation shell of
water is exchanged for the polar groups responsible for hydrogen bond
interactions in the binding site of the protein. As in the cases of MBP
and other periplasmic binding proteins, AlgQ2 has two globular domains
connected by a flexible hinge, and in the ligand-bound structure, the
ligand is buried deep within the cleft between the two domains. In the
absence of a ligand, the cleft between the domains is more open and
exposed to the solvent.
However, there are significant differences in protein-sugar
interactions between AlgQ2 and MBP. The S1 subsite seems to be the most
important one for the binding of alginate in AlgQ2, and it may be
specific for mannuronate in this structure. The conformations of MM and
GG blocks are known to be linear and helical, respectively (24). Based
on the conformation of alginate, mannuronate is favorable at the S1
subsite. Although in other periplasmic binding proteins, a reducing
terminal sugar binds at the S1 subsite, AlgQ2 binds a nonreducing
terminal sugar at the S1 subsite (22, 26-28). Furthermore, AlgQ2
interacts strongly with a nonreducing terminal sugar, although a
glucose positioned at the S2 subsite participates in the greatest
number of hydrogen bonds and van der Waals contacts in MBP complexed
with an oligosaccharide (28). Compared with other periplasmic binding
proteins, the sugar binding cleft of AlgQ2 extends farther toward the
core of the protein. As judged on computational simulation, at least
hexasaccharide can be bound without exposure to the solvent.
A ligand-induced conformational change is believed to be crucial for
the function of a periplasmic binding protein in both transport and
chemotactic processes. Such a conformational change generates the
appropriate stereochemistry for specific interaction of the liganded
protein with membrane-bound protein components, in preference to the
unliganded form, thus initiating the translocation process.
On going from the open cleft, unliganded structure to the closed
structure, there is a concerted shift of the Glu396 side
chain, which moves up into the cleft as a result of sugar binding (Fig.
5). This ligand-induced movement of Glu396 may be the
triggering mechanism for the motion that enables the other domain to
participate in ligand binding and ultimately to engulf the bound
tetrasaccharide. The major driving force for the hinge closing in AlgQ2
is probably the result of exclusion of a water molecule from the
binding site. The shift in the equilibrium on sugar binding may also be
enhanced by the interaction of the sugar with Glu396, which
results in the perturbation of the hinge favoring the closed form.
Because Glu396 is hydrogen bonded to M1-O3 in the S1
subsite in the closed form, it also may reflect the importance of S1
subsite for binding alginate.
Two or three water molecules function as hinge water in the ABP and RBP
(29, 30) belonging to group I judging from transitions from domain to
domain. In ABP and RBP, hinge water molecules play an important role in
hinge motion and are also involved in the stabilization of each form.
However, in AlgQ2, only one water molecule (WAT715) is responsible for
hinge motion, and the loss of WAT715 is likely to cause dynamic hinge
movements. Because this is the first example of a hinge water molecule
of group II binding proteins, the water-mediated hinge motion observed
in AlgQ2 should facilitate the clarification of the conformational changes in group II binding proteins.
This is the first reported description of the structural and functional
relationship of a periplasmic binding protein of a bacterial ABC
transporter which binds macromolecules. Further analyses of the
structural features of AlgQ2 complexed with oligo- and polysaccharides
and comparison with AlgQ1, which resembles AlgQ2 on alignment, should
provide new insights into the mode of binding an acidic polysaccharide.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
-D-mannuronate (M)
and its C5 epimer,
-L-glucuronate (G) (5). There are
block structures that have been shown to cause the gelation and
viscosity of the alginate produced by brown seaweed or certain kinds of
bacteria (6). The block structures are usually arranged as
homopolymeric poly-
-D-mannuronates (MM blocks),
homopolymeric poly-
-L-glucuronates (GG blocks), or
heteropolymeric saccharides (MG or GM blocks) within the alginate
molecule (7).
6-10
7 M (8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
= 0.9 Å, using an Oxford PX210 CCD detector system
at beam line BL44XU (Beamline for Macromolecule Assembles, Institute
for Protein Research, Osaka University) at SPring-8 (Hyogo, Japan). The
data collection was carried out at the temperature of liquid nitrogen.
A complete data set was recorded for a single crystal with an exposure
time of 6 s for 1° oscillations. The collected images were
processed with program D*Trek (11) and the CCP4 truncate program
(12).
Fc and 2 Fo
Fc maps were used to locate the correct
model. Several rounds of conjugate gradient minimization refinement and
B factor refinement, followed by manual model building, were
carried out to improve the model by increasing the data to 1.6-Å
resolution. Water molecules were incorporated when the difference in
density was more than 3.0
above the mean and the
2 Fo
Fc map
showed a density of more than 1.0
.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
= 107.53°, and the
solvent content was 45.0% assuming two molecules (molecules A and
B)/asymmetric unit. The results of x-ray data collection and the
refinement statistics are summarized in Table I. The structure of holo-AlgQ2 was
determined by the molecular replacement method, using apo-AlgQ2 (9) as
a search model, and refined at 1.6-Å resolution. All of the
polypeptide chain sequence could be well traced, and the electron
densities of the main and side chains were generally very well defined
on the 2 Fo
Fc
map. The final R factor was 19.0% for 145,984 data points in the 61.6-1.6-Å resolution range (99.0% completeness). The
Rfree value calculated for the randomly
separated 10% data was 21.1%. Based on the theoretical curves in the
plot calculated according to Luzzati (19), the absolute positional
error was estimated to be close to 0.18 Å between 5.0- and 1.6-Å
resolution. Judging from the results of Ramachandran plot analysis
(20), in which the stereochemical correctness of the backbone structure
is indicated by the (
,
) torsion angles, we found that most of
the non-glycine residues lie within the most favored regions, the
exception being Lys251 (molecule A:
= 65.0°,
=
137.4°; and molecule B:
= 62.5°,
=
139.9°), which is present in a generously allowed region. Lys251 is located next to the terminus of a helix (H12)
(9).
Statistics for data collection and refinement
backbone trace of holo-AlgQ2 together with the bound
trisaccharide and a calcium ion. The final model comprises 984 amino
acid residues, 2 tetrasaccharides, 2 calcium ions, and 821 water
molecules/asymmetric unit.
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Fig. 1.
a, stereo view of a C backbone
trace of holo-AlgQ2 (blue) showing the locations of the
binding sites for a calcium ion (yellow) and the
tetrasaccharide (red). N, N-terminal end;
C, C-terminal end. This figure was prepared using MOLSCRIPT
(16) and RASTER3D (17). b, stereo view of a C
backbone
trace of the superpositioned structures of holo-AlgQ2 (blue)
and apo-AlgQ2 (yellow). N and C denote
the two terminal ends. The apparent rotation axis is shown as a
dashed line. This figure was prepared using MOLSCRIPT (16)
and RASTER3D (17).
/
-structure. The tetrasaccharide is bound
in the deep cleft between the domains. Both domains exhibit a similar
arrangement of the elements of the secondary structure and can be
divided further into two parts (N1 and N2 domains, and C1 and C2
domains) (9).
atoms that are within a
distance of 2.0 Å of one another is 0.67 Å (295 C
atoms). These
values are 0.42 Å for the N domain (224 C
atoms) and 0.47 Å for
the C domain (264 C
atoms). To characterize the conformational
changes that occur upon domain opening and closing, the domain motion
was considered separately as rotation and translation (23). The N
domains of apo and holo forms were superimposed, and the rotation and
translation required to superimpose the C domains were determined to be
30° and 0.5 Å, respectively (Fig. 1b).
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Fig. 2.
Molecular surface models of apo- and
holo-AlgQ2. Left, apo-AlgQ2; right,
holo-AlgQ2. The structures are represented as white molecular surface
models of AlgQ2. Basic and aromatic amino acid residues are colored
magenta (Lys, Arg, His); Trp, Tyr, and Phe are
yellow. The tetrasaccharide molecule is shown as a
thick blue line. This figure was prepared using GRASP
(18).
M1-M2-G3-M4, from the nonreducing end, where
M, M, and G denote
unsaturated and saturated
-D-mannuronate and saturated
-L-glucuronate, respectively (Fig.
3), and the sugar binding sites,
corresponding to the alginate residues, are designated as S1, S2, S3,
and S4, respectively (Fig. 3b). Each alginate residue of the
bound tetrasaccharide (
M1-M2-G3-M4) was in the
4C1 (M2, M4) or 1C4
-pyranosid form (G3). The sugar difference density of S1 only fits
unsaturated mannuronate. The tetrasaccharide used for cocrystallization with AlgQ2 was prepared through depolymerization of HMW alginate with
alginate lyase A1-III (10). Depolymerization produces a C4-C5 double
bond at the nonreducing terminal mannuronate residues of the products,
which are mixtures of MM, MG, and GM blocks (24). However, we found
M1-M2-G3-M4 in both molecules A and B, which may indicate that
M1-M2-G3-M4 was the predominant species of tetrasaccharide obtained
in the present experiment. The bound tetrasaccharide adopts a linear
shape (Fig. 3a). The torsion angles of the glycosidic bonds,
defined as O5'-C1'-O4-C4 (
) and C1'-O4-C4-C5 (
), between
M1
and M2, M2 and G3, and G3 and M4 of each molecule, are shown in Table
II. The torsion angles between
M1 and
M2 in both molecules are in the lowest energy region of the isoenergy map. According to the report by Braccini et al. (25), the
adiabatic maps of glucuronic and mannuronic acid dimers are almost
identical, and the energy minima are similar, with identical (
,
)
values and comparable relative energies. The torsion angles between M2 and G3, and G3 and M4 are also in the lowest energy region of the
isoenergy maps of both mannuronic and glucuronic acid dimers. These
results suggest that bound tetrasaccharides are in a stabilized conformation.
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Fig. 3.
a, stereo view of a
2 Fo Fc map of
the tetrasaccharide. The 2 Fo
Fc map contoured with 1.0
and a
tetrasaccharide molecule are shown as pink thin lines and
blue thick lines, respectively. This figure was
prepared using TURBO-FRODO. b, subsites for alginate binding
by AlgQ2.
Dihedral angles around the glycosidic linkage of the tetrasaccharide
-anomeric, and M4-O1 is wholly
axial (Fig. 3a). No other electron density is present in the
binding site. Hydrogen bonds are formed between M4-O1 and two water
molecules (molecule A, WAT36 and WAT325; molecule B, WAT559 and
WAT697). WAT36 is hydrogen-bonded to Asp74-OD1, -OD2, and
Gln55-NE2. This preferential binding of the
-anomer to
AlgQ2 is because of the water-bridged hydrogen bonds between axial
M4-O1 and the amino acid residues.
M1, 10;
M2, 8; G3, 6; and M4, 7). The number of direct hydrogen bonds between
AlgQ2 and the tetrasaccharide is 14 (
M1, 8; M2, 3; G3, 2; and M4,
1), and the number of associated water molecules is 17 (
M1, 2; M2,
5; G3, 4; and M4, 6).
M1 participates in the greatest number of
hydrogen bonds, followed by M2, G3, and M4. This suggests that the
nonreducing end of a sugar is more heavily involved than the reducing
end of the sugar.
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Fig. 4.
Stereo view of the binding site of AlgQ2 with
the bound tetrasaccharide. The hydrogen bonds (broken
lines) and stacking interactions between the tetrasaccharide and
AlgQ2 are shown. The aromatic residues (yellow), basic
residues (pink), acidic residues (cyan), and
nonpolar residues (green) are shown. This figure was
prepared using GRASP (18).
Hydrogen bond interactions between the tetrasaccharide and AlgQ2
M1 in
the apo form (9) and form the same hydrogen bond with the protein.
Water molecules occupying the positions of hydroxyl groups of
carbohydrates to preserve the hydrogen bonding interactions in the
sugar binding region have also been observed in the structure of
glucoside-related enzymes. Furthermore, a typical characteristic of
uronic acids is that C6 is oxidized to carboxyl acid. Thus, these
residues (O61 and O62) may confer the specificity to AlgQ2.
M1 (Ser273,
Asn375, Tyr395, and Glu396) are
located in helices, all residues binding with M2 (Tyr129,
Arg186, and Trp270) in
-sheets, and all
residues binding with G3 and M4 (His187,
Arg313, and Asp21) in loops. In MBP, most
residues binding with maltose are located in loops of the N domain, and
its localization may participate in the hinge bending motion (27). But
in AlgQ2, most of these residues are in the helices of both domains and
-sheets, with a few in loops.
M1, 23; M2, 7; G3, 6; and M4, 3). In holo-AlgQ2, almost all the
nonpolar-nonpolar contacts between AlgQ2 and the tetrasaccharide are
mainly through aromatic side chains (Trp270,
Trp399, and Tyr129). Indeed, the close
proximity of aromatic side chains to bind a tetrasaccharide is a
recurring feature of proteins/enzymes that bind tetrasaccharides. The
structure of AlgQ2 is notable among those of proteins/enzymes in that
AlgQ2 binds saccharides because of the great abundance of aromatic
residues, 16 of which are located in or near the binding site groove.
In contrast to most hydrogen-bonding residues located in the N domain,
more stacking aromatic residues are found in the C domain. In
particular, the aromatic Trp270 residue in the C domain
exhibits the most favorable stacking interactions with
M1. Such
stacking interactions between a sugar and aromatic side chains are
quite common in protein-carbohydrate complexes (28). Of special
significance regarding AlgQ2 is the finding that some of the van der
Waals contacts are confined to a cluster of nonpolar atoms within the
sugar. In the alginate molecule, the disposition of both the axial OH2
and equatorial OH3 of
M1 on the hydrophilic side of the ring creates
a cluster of nonpolar atoms (C3, C4, and C5) on the opposite side. This nonpolar cluster is stacked with Trp270 (Fig. 4). Because
MM blocks may form a cluster of nonpolar atoms within a sugar most
easily,
MM and MM blocks seem to be bound at the S1 and S2 subsites.
In other periplasmic binding proteins, the aromatic residues are
partially stacked or face to face with the pyranose ring. As in MBP,
aromatic residues in the C domain of AlgQ2 play an important role in
the interaction with HMW alginate.
C-C contacts between the tetrasaccharide and AlgQ2
M1 position
by computational simulation. If saturated mannuronate is positioned at
the
M1 position, O4 interacts with OE1 and OE2 of Glu396
at a distance of about 2.8-2.9 Å. When glucuronate is present instead
of
M1, glucuronate cannot form a C-C contact with Trp270
or other aromatic residues because both the axial OH2 and equatorial OH3 of
M1 are displaced to the hydrophilic side. This suggests that
the S1 subsite may bind saturated and unsaturated mannuronate acids selectively.
-sheet in N1 domain; SC,
-sheet in C1 domain; H,
-helix; these notations have been defined
previously (9)).
M1-O3. As a consequence of
this change, the torsion angles of Arg309 greatly increased
(
= 45°,
= 33°), and hinge bending motion occurred in L-SC4:SA3. As a result of this movement, the indirect water-mediated hydrogen bond between Gly133 (L-SA1:SC3) and
Gln310 (L-SC4:SA3) via a water molecule (WAT715) is lost,
and both the
and
angles of Gly133 and
Gln310 change greatly (Gly133,
=
49° and
=
27°; Gln310,
= 37° and
= 41°). In the closed form, Gln310
forms hydrogen bonds with residues of the C domain (Fig. 5 and Table
V). In particular, Asp206 and Asn207 (L-H9:SD1)
are in the loop connecting the C1 and C2 domains. These interactions
may stabilize AlgQ2 in the closed form.
View larger version (59K):
[in a new window]
Fig. 5.
a, hinge region of apo-AlgQ2 (open form,
left) and holo-AlgQ2 (closed form, right).
b, their models. Residues in L-SA1:SC3 are
yellow; in L-SC4:SA3, red; in L-H19:H20,
orange; in H19, cyan; and in L-H9:SD1,
pink; and the water molecule (WAT715), in green.
a was prepared using GRASP (18).
Hydrogen bonds between the N and C domains of apo- and holo-AlgQ2
M1, a
conformational change occurs in L-SC4:SA3, which interacts with
Glu396 through hydrogen bonds. As a result of this
conformational change, the association via a water molecule (WAT715)
between L-SA1:SC3 and L-SC4:SA3 is lost, and both loops undergo a
dynamic hinge bending motion. A water molecule (WAT715) and the
interaction between Glu396 and L-H9:SD1 via L-SC4:SA3 are
important for the hinge motion of AlgQ2.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to K. M., B. M., and W. H.) and by a grant from the Japan Society for the Promotion of Science (to Y. M.). A part of this work was supported by the Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan. The x-ray diffraction experiments were performed at beamline BL44XU at SPring-8 (Proposal C01A44XU-7136-N).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1J1N) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-774-38-3766; Fax: 81-774-38-3767; E-mail:
murata@food2.food.kyoto-u.ac.jp.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M209932200
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ABBREVIATIONS |
---|
The abbreviations used are:
ABC, ATP-binding
cassette;
HMW, high molecular weight;
ABP, L-arabinose-binding protein;
G, -L-glucuronate;
GBP, galactose-binding protein;
GG
block, homopolymeric poly-
-L-glucuronate;
GM block, heteropolymeric saccharide;
M,
-D-mannuronate;
M, unsaturated
-D-mannuronate;
MBP, maltodextrin/maltose-binding protein;
MG block, heteropolymeric
saccharide;
MM block, homopolymeric poly-
-D-mannuronate;
RBP, ribose-binding protein;
WAT, water molecule.
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REFERENCES |
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