(Received for publication, September 13, 1995)
From the
The structural basis of carbohydrate recognition by rat liver
mannose-binding protein (MBP-C) has been explored by determining the
three-dimensional structure of the C-type carbohydrate-recognition
domain (CRD) of MBP-C using x-ray crystallography. The structure was
solved by molecular replacement using rat serum mannose-binding protein
(MBP-A) as a search model and was refined to maximum Bragg spacings of
1.7 Å. Despite their almost identical folds, the dimeric
structures formed by the two MBP CRDs differ dramatically. Complexes of
MBP-C with methyl glycosides of mannose, N-acetylglucosamine,
and fucose were prepared by soaking MBP-C crystals in solutions
containing these sugars. Surprisingly, the pyranose ring of mannose is
rotated 180° relative to the orientation observed previously in
MBP-A, but the local interactions between sugar and protein are
preserved. For each of the bound sugars, vicinal, equatorial hydroxyl
groups equivalent to the 3- and 4-OH groups of mannose directly
coordinate Ca and form hydrogen bonds with residues
also serving as Ca
ligands. Few interactions are
observed between other parts of the sugar and the protein. A complex
formed between free galactose and MBP-C reveals a similar mode of
binding, with the anomeric hydroxyl group serving as one of the
Ca
ligands. A second binding site for mannose has
also been observed in one of two copies in the asymmetric unit at a
sugar concentration of 1.3 M. These structures explain how
MBPs recognize a wide range of monosaccharides and suggest how fine
specificity differences between MBP-A and MBP-C may be achieved.
Mannose-binding protein C (MBP-C) ()is a C-type
(Ca
-dependent) lectin isolated from rat
liver(1) . MBP-C belongs to the collectin group of C-type
lectins, which also includes the serum mannose-binding proteins and
pulmonary surfactant apoproteins A and D(2) . The collectins
share a common arrangement of structural domains: a cysteine-rich
domain at the amino terminus is followed by a collagenous domain, an
oligomerization domain, and a COOH-terminal carbohydrate recognition
domain (CRD). Individual polypeptides assemble into homotrimers, which
further associate into larger oligomers. Serum MBPs, such as MBP-A in
rat and human MBP, consist of hexamers of trimeric building blocks (M
650,000). Serum MBPs function in
antibody-independent host defense against pathogens by binding avidly
to carbohydrate structures on foreign cell surfaces and inducing
opsonization and complement-mediated cell
lysis(3, 4, 5) . MBP-C is a smaller oligomer
than serum MBPs, and probably consists of two associated trimers (M
200,000)(6) . Although the
function of MBP-C is not known, the structural similarities between it
and other collectins suggest that MBP-C may also play a part in host
defense. Localization of MBP-C to the liver suggests that it may also
have roles in cell-cell interactions or glycoprotein
trafficking(7) .
Consistent with the requirement that they recognize a variety of cell surfaces, MBPs A and C bind to a number of different monosaccharides containing vicinal, equatorial hydroxyl groups such as those found at the 3 and 4 positions of mannose (Man), including N-acetylglucosamine (GlcNAc) and fucose (Fuc)(8) . In contrast, MBPs A and C show minimal affinity for sugars that lack hydroxyl groups in this arrangement, such as galactosides and sialic acids. In addition to its characteristic monosaccharide specificity, MBP-C shows finer specificity differences for binding to complex N-linked oligosaccharides that contain Man, GlcNAc, and Fuc (9) . MBP-C binds to a wide range of ligands that contain the trimannosyl core common to N-linked oligosaccharides, but the specific oligosaccharide structures recognized by MBP-C are not known. It is clear, however, that MBPs A and C have different binding specificities for complex oligosaccharides and other multivalent ligands, including neoglycoproteins(9) . For example, MBP-A binds GlcNAc-BSA much more tightly than MBP-C, even though the two proteins bind comparably to free GlcNAc.
Sugar
binding by MBPs A and C is mediated by the COOH-terminal CRD. Related
CRDs of 115-130 amino acids are present in all C-type lectins.
Structure-based sequence alignments(10) , as well as the
crystal structures of two distantly related C-type
lectins(10, 11) , suggest that the overall structure
of the CRD is very similar in all C-type lectins. The high-resolution
crystal structure of a complex between a high-mannose oligosaccharide
and the MBP-A CRD (12) shows that Man binds at a conserved
Ca site, designated site 2 (10, 12) , through vicinal, equatorial 3- and 4-OH
groups that form coordination bonds with the Ca
and
hydrogen bonds with amino acid side chains that also serve as
Ca
site 2 ligands. Sequence alignment, mutagenesis,
and crystallographic studies suggest that this binding site is
well-conserved among C-type lectins, and that the sequence of amino
acids around this site determines binding
specificity(11, 13, 14, 15, 16, 17, 18, 19) .
Modeling suggests that other sugars known to bind to MBPs can bind in a
similar manner, and the proposed mode of Fuc binding to MBP (12) has been used for modeling selectin-ligand
interactions(11, 19) .
Here we describe the
structure of the MBP-C CRD, alone and complexed with a series of
monosaccharide ligands. Although the structure of the MBP-C protomer is
very similar to that of MBP-A, the arrangement of protomers in the
dimers formed by isolated CRDs differs drastically. The crystals formed
by the MBP-C CRD diffract to high resolution and, unlike previous
crystals of the MBP-A CRD(20) , do not require sugar ligand for
growth, thus providing a means to study a variety of sugar-protein
complexes. The structures of MBP-C complexed with five different
monosaccharides reveal how different sugars are recognized by MBPs and
other C-type lectins. The local bonding scheme between protein and
sugar is similar to that observed in the oligosaccharideMBP-A
complex, but mannose is bound in a different orientation from that
observed in MBP-A.
Figure 1:
MBP-C domain organization. The domain
organization of natural MBP-C, the expressed fragment, and the
subtilisin fragment are summarized. The sequence of the expressed
protein around the subtilisin cleavage sites is presented along with
the proportion of fragments produced by subtilisin cleavage at
different sites, as determined by NH-terminal sequence
analysis. The complete sequences of sub-MBP-A and sub-MBP-C are also
aligned, with vertical lines denoting identical residues and dots above the sequences marking every 10th
residue.
Figure 2:
Subtilisin digestion of MBP-C. Aliquots of
purified, bacterially expressed MBP-C were digested with 0, 5, 10, 20,
40, and 80 µg/ml subtilisin in 1.25 M NaCl, 25 mM Tris-Cl (pH 7.8), and 25 mM CaCl at 37 °C
for 1 h. Following electrophoresis on a SDS-polyacrylamide gel (17.5%),
digest products were detected by staining with Coomassie Blue. The
sizes of molecular weight standards (in kDa) are shown at the left.
Data were measured on an R-AXIS
IIC imaging-plate detector (Rigaku), using CuK radiation from a rotating anode (Rigaku; 50 kV, 90 mA, graphite
monochromator, 0.3-mm collimator) at a crystal-to-detector distance of
85 mm. Lorentz polarization-corrected integrated intensities were
obtained using DENZO(21) , and redundant measurements were
scaled and merged with SCALEPACK(21) . Each data set was put on
a quasi-absolute scale using TRUNCATE (22) and scaled to the
data from the sugar-free crystal using RSTATS(22) . Data
processing statistics are presented in Table 1. X-PLOR (23) was used for molecular replacement, reciprocal space
refinement, and electron density map calculations. Model building was
performed using O (24) . Throughout the course of refinement,
regions where the model fit the electron density poorly or had poor
geometry were omitted and rebuilt according to simulated annealing omit
maps(25) . The geometry of the model was monitored during
refinement using X-PLOR and PROCHECK(26) .
Sequence analysis of purified
sub-MBP-C indicates some heterogeneity in the NH-terminal
sequence. Relative amounts of different fragments are indicated in Fig. 1. The unique site of cleavage in MBP-A corresponds to
Val
of MBP-C, suggesting that a similar region of the two
proteins is exposed to digestion. The oligomeric state of sub-MBP-C was
investigated by gel filtration chromatography and chemical
cross-linking. The fragment elutes with an apparent molecular weight of
21 kDa from a gel filtration column (data not shown). Since the
polypeptide molecular weight is 13 kDa, the elution position suggests
that the fragment is dimeric. This conclusion is supported by
cross-linking data (Fig. 3), in which the fragment containing
neck and CRD forms trimers, while the CRD alone forms only dimers.
Thus, like the CRD of MBP-A, the MBP-C CRD appears to undergo a
rearrangement in oligomer geometry upon removal of the adjacent neck.
Figure 3: Chemical cross-linking of MBP-C. Aliquots of binding domains (left sub-MBP-C; right bacterially expressed MBP-C) were treated with 0, 1.5, 3, and 6 mM bis(sulfosuccinimidyl)-suberate for 1 h at 22 °C. Following electrophoresis on a SDS-polyacrylamide gel (15%), cross-linked complexes were detected by staining with Coomassie Blue.
Crystals of sub-MBP-C were grown in the presence of Ca at neutral pH. Precession photography revealed the space group to
be P2
2
2
. Based upon the unit cell
dimensions, the calculated molecular weight of a protomer (13 kDa), and
a partial specific volume of 0.73 g/cm
for the protein, the
crystal would contain either 77, 54, 31, or 8% solvent if it contained
one, two, three, or four protomers/asymmetric unit. Because space group
P2
2
2
does not contain a pure 2-fold
rotation axis, if a 2-fold axis exists in the sub-MBP-C dimer, the
asymmetric unit would most likely consist of a single dimer. If the
dimer lacks a 2-fold axis, there could be from one to three
protomers/asymmetric unit. Self-rotation functions calculated from the
data used for molecular replacement did not reveal the presence of a
noncrystallographic rotation axis.
When a single protomer was positioned according to either of the two translation function solutions, the R-factor was unusually high (0.62). When the second protomer was positioned according to the repeat shown in the native Patterson and translation functions, however, the R-factor was 0.48, a value more typical of a correct molecular replacement solution. This behavior arises because the interference function generated by the repeat along x cannot be modeled with a single protomer. Visual inspection indicated that the molecular packing in the crystal was reasonable. Finally, the model was refined first as a single rigid body and then with individual protomers as separate rigid bodies, yielding an R-factor of 0.41 for data between 10 and 2.8 Å.
Initial rounds of map calculation and refinement used data from 10
to 2.8 Å. Calcium ions, side chains not present in the search
model, and main chain positions that differed significantly from the
search model were manually positioned using 2F - F
and F
- F
electron density maps. After a round of
positional refinement, resolution limits were changed to the range of
5-2.5 Å, and resolution-dependent weighting was introduced.
A round of simulated annealing refinement at 4000 K was followed by
manual model building. Resolution limits were then extended to
5-2.3 Å, and individual isotropic temperature factors were
reset (22 Å
for main chain atoms and 26 Å
for side chains) and refined. Additional rounds of positional and
temperature-factor refinement were carried out against data from
5-2.0 Å, and solvent molecules were added. Finally, atomic
positions and temperature factors were refined against data from
10-1.7 Å, when alternate side chain conformations and
weaker solvent sites were added to the model. The final temperature
factors and occupancies of solvent atoms were refined by resetting the
temperature factor to a value equal to the average of all
hydrogen-bonded neighbors plus 5 Å
and by iteratively
refining occupancy and temperature factor. Only solvent atoms visible
at the 1.0
contour of 2F
- F
maps, and which were capable of making at least
one hydrogen bond with either a protein atom or another solvent atom,
were retained in the model. A representative portion of the final
electron density map is shown in Fig. 4.
Figure 4:
Dimer
interface of MBP-C. Stereo view of electron density (contoured at 1.0
) superimposed on the final model of unliganded MBP-C. The number
preceding the residue identifier indicates which protomer the residue
is from. The view is approximately down the 2-fold noncrystallographic
symmetry axis, showing the interaction between Leu
of one
protomer and the pocket formed by Leu
, Leu
,
and Cys
(disulfide-bonded to Cys
) of the
other protomer. The figure was prepared using
O(24) .
The final model
contains all of sub-MBP-C except residues 111-115 at the NH terminus and residue 226 at the COOH terminus. Electron density
is weak or absent for the following surface side chains: in protomer 1,
Tyr
, Lys
, Arg
, and
Arg
; in protomer 2, Tyr
, Gln
,
Arg
, and Glu
. The side chains of five
residues (Met
in both protomers, Arg
in
protomer 1, and Ser
and Thr
in protomer 2)
were modeled in two conformations. All main chain torsion angles fall
within allowed regions of the Ramachandran plot. Model geometry and
temperature-factor statistics are summarized in Table 2.
Coordinate error was assessed by several methods. Luzzati analysis (30) suggests that the coordinate error is in the range
0.15-0.25 A, although the behavior of the R-factor with scattering angle does not follow the theoretical
curves very well (not shown). As an alternative estimate of coordinate
precision, the final structure was subject to two simulated annealing
runs at 4000 K followed by minimization. Superposition of the
structures gave an overall root mean square deviation of 0.30 A for atoms whose temperature factors are less
than 50 Å
. This is most likely an overestimate, since
the molecular dynamics trajectory is not well constrained by the data
for surface residues. Finally, superposition of the main chain atoms of
the two independent copies in the asymmetric unit gives a root mean
square deviation of 0.24 Å if regions clearly different due to
lattice contacts (see below) are removed. Thus, the coordinate error
appears to be on the order of 0.20-0.25 Å.
Figure 5:
Structure of MBP-C and MBP-A dimers. Main
chain atoms in protomer 1 of the MBP-A dimer (dashed line)
were superimposed onto the main chain atoms of protomer 1 of the MBP-C
dimer (solid line) by least squares minimization. The
differences in dimer structure are emphasized by the very different
positions of the second protomers in MBP-A versus MBP-C.
Calcium ions (black for MBP-C and white for MBP-A)
are numbered. The asterisk marks the location of the second
ligand binding site see at 1.3 M -Me-Man. The figure was
prepared using MOLSCRIPT(49) .
The asymmetric unit of
the MBP-C crystal consists of a noncrystallographic dimer that
corresponds to the dimer observed in solution (Fig. 5). When one
protomer is superimposed on the other, the root mean square deviation
of all main chain atoms is 0.32 Å. Significant differences
between the two protomers are restricted to residues near different
lattice contacts in the two independent copies of the protomer:
residues 153-154 (), 177-181 (loop 3), and
195-196 (loop 4) (see Weis et al.(10) for
secondary structure assignments). The amount of buried surface area in
the dimer is roughly 650 Å
/protomer, which is
one-eighth of the total surface area of the protomer. The amount of
buried surface is comparable with that seen in the MBP-A dimer (10) as well as other protein dimers(31) .
A
noteworthy difference between MBP-A and MBP-C is the lack of the third
Ca site seen in both rat MBP-A(12, 32) and human MBP(33) . In MBP-A, three of the
Ca
site 3 ligands come from the side chains of
Glu
and Asp
, which also serve as ligands
for Ca
site 1; Asp
provides two of the
ligating oxygens (see Fig. 2b of (12) ). In
MBP-C, Asn
is equivalent to Asp
of MBP-A,
and apparently cannot provide the requisite ligands for
Ca
site 3. Based on the fact that carbohydrate
binding and adoption of a protease-resistant conformation by MBP-A
requires two Ca
(20) , it has been argued that
Ca
site 3 is adventitious and is observed due to the
high concentrations of Ca
used for
crystallization(12, 32) . The lack of this site in
MBP-C confirms that it is not a conserved feature of MBPs.
The starting model for
refining different sugar-protein complexes was derived from the fully
refined model for the unliganded protein. The positions of 246 water
molecules were independently determined for the unliganded protein and
the -Me-Man and
-O-methyl-N-acetylglucosamine (
-Me-GlcNAc)
complexes. For all other complexes, these solvent positions were
transferred, refined, and carefully checked against against
2F
- F
electron density
maps. For each complex, the starting model without sugar was refined
against data from 10 Å to the highest resolution measured
(1.7-1.9 Å) (Table 1) before an F
- F
difference electron density map
was calculated. Unbiased, positive F
- F
difference electron density defined the entire
sugar molecule bound at Ca
site 2; however, the
aglycon was not visible in any case except for
-O-methyl
fucoside (
-Me-Fuc) and one copy of
-Me-Man. A model for the
bound sugar with regular geometry could be placed unambiguously into
positive F
- F
difference electron density, and water molecules near the sugar
were then added. The model was subject to further rounds of positional
and temperature factor refinement, and the temperature factors and
occupancies of the water molecules were iteratively refined as
described for the unliganded protein. Representative electron density
for a bound sugar at Ca
site 2 is shown in Fig. 6c, and model geometry and temperature-factor
statistics are presented in Table 2.
Figure 6:
Displacement of water upon Man binding.
Electron density (contoured at 1.0 ) from unliganded MBP-C
superimposed on models for unliganded MBP-C (a) and the model
of
-Me-Man bound to MBP-C (b). In c, the
electron density (contoured at 1.0
) for the complex of
-Me-Man and MBP-C is superimposed on the model for that complex.
The 3- and 4-OH of the bound mannoside superimpose on the water
molecules bound to the unliganded protein. The figure was prepared
using O(24) .
Figure 7:
Structures of sugars bound to MBP-C. The
structures of five different sugars bound to MBP-C are shown along with
the structure of the terminal mannose residue bound to
MBP-A(12) . The remaining portion of the oligosaccharide bound
to MBP-A has been omitted for clarity. The ligands have been
superimposed and are presented in a common view. Carbon, nitrogen, and
oxygen atoms are shown as white, gray, and black spheres, respectively. Ca 2 is shown as a larger gray sphere. Carbon atoms of the bound sugars are
numbered. Long-dashed lines denote coordination bonds with
Ca
, medium-dashed lines denote hydrogen
bonds, and short-dashed lines denote van der Waals'
contacts. Note that the methyl aglycon is visible only in
-Me-Fuc
and in one of the two copies of
-Me-Man (not shown). The figure
was prepared using MOLSCRIPT(49) .
Figure 8:
Van der Waals' contacts between
MBP-A or MBP-C and Man. View of the interactions between the terminal
Man residue of a bound oligosaccharide and MBP-A (a) (12) and a view of the interactions between -Me-Man and
MBP-C (b). In a, the remaining portion of the
oligosaccharide has been omitted for clarity. The view is roughly
90° away from the view presented in Fig. 7. Atoms are shaded
as in Fig. 7. Van der Waals' contacts are denoted by dashed lines. The figure was prepared using
MOLSCRIPT(49) .
Surprisingly, the positions of the 3- and 4-OH groups of
-Me-Man are reversed in MBP-C when compared with MBP-A (Fig. 7)(12) . A dyad axis relates the vicinal,
equatorial hydroxyl groups and the carbon atoms to which they are
directly bonded, so the local geometry of hydrogen and coordination
bonds is the same for both orientations. Modeling indicates that the
binding sites of MBPs A and C would allow Man to bind in either
orientation, but the electron density clearly shows that Man is bound
in a single orientation in both cases (Fig. 6)(12) .
Moreover, in both dimer structures there are two independent copies in
the crystallographic asymmetric unit, which argues against effects of
lattice environment on the orientation. In the case of MBP-A, however,
the orientation of Man is known from the structure of a cocrystal of a
high mannose oligosaccharide and the sub-MBP-A dimer(12) . The
oligosaccharide cross-links the crystal such that the terminal Man
residues of two nonequivalent branches (
-(1,2) and
-(1,3))
are bound to the two independent copies. It is possible that other
sugars, including other mannosides, bind to MBP-A in the orientation
observed in MBP-C.
The paucity of interactions between the protein
and -Me-Man reflects the openness of the sugar-binding site, which
can be described as a shallow trough. As shown in Fig. 9, one
edge of this trough is formed by Val
. Binding of
-Me-Man to MBP-C buries approximately 130 Å
(40%) of sugar and 80 Å
of protein
solvent-accessible surface area (calculated with a probe radius of 1.4
Å(34) ). For comparison, binding of the terminal Man to
MBP-A buries 160 Å
of sugar and 90 Å
of protein(12) . Interestingly, the less polar surface of
-Me-Man faces Val
( Fig. 8and Fig. 9).
Although this surface is largely exposed to solvent, no ordered water
molecules are visible near it. In contrast, an extensive network of
water molecules extends along the other surface of the sugar. Except
for six water sites closest to the sugar itself, the network of water
sites in this binding trough is not disrupted by binding of the sugar.
Figure 9:
Water structure in the MBP-C-Man complex.
Stereo view of protein residues 190-198 and 210-213 of
MBP-C, -Me-Man, and all water molecules within 5.0 Å of a
sugar atom. Atoms are shaded as in Fig. 7. Long-dashed lines denote coordination bonds with Ca
, medium-dashed lines denote hydrogen bonds, and short-dashed lines denote van der Waals' contacts. All
protein-water, sugar-water, and water-water hydrogen bonds are shown.
The figure was prepared using
MOLSCRIPT(49) .
Superposition of liganded and unliganded structures, as well as
isomorphous difference Fourier maps, reveal that the binding of
-Me-Man does not detectably affect the conformation of the
protein. Moreover, the temperature factors of residues in the
sugar-binding site do not change significantly upon sugar binding. The
most notable effect of sugar binding is the displacement of two water
molecules, which form the same set of coordination and hydrogen bonds
as described for the sugar (Fig. 6, Table 4). The sugar
hydroxyl groups superimpose perfectly over the displaced water
molecules, preserving the 8-fold coordination of the calcium ion.
The significantly weaker
binding of free galactose relative to the other sugars studied here,
despite the same number of contacts formed with the protein, may be due
to the unique electronic properties of the anomeric oxygen that make it
a relatively poor hydrogen bond acceptor (38) . This property
may also make the coordination bond between the anomeric oxygen and
Ca relatively weak. In addition, since only the
-anomer of Gal has the correct disposition of 1- and 2-OH for
binding, and the ratio of
- to
-Gal is approximately 1:2 in
solution(39) , the apparent affinity for Gal is diminished by a
factor of 1.7.
Aside from the interactions with Ca and Ca
ligands, the only other interaction
between sugar and protein common to all complexes is the van der
Waals' contact between C-3 of the pyranose ring and the side
chain of Val
(Fig. 8). In all complexes, this
interaction is slightly different in the two different copies in the
asymmetric unit, although each protomer makes the same interactions
with each of the bound sugars, as noted above for the
-Me-Man
complex (Table 4). The small number of interactions between sugar
and protein attests to the importance of the interactions between
(+)-syn-clinal hydroxyl groups on bound sugars and
Ca
site 2, as well as the openness of the sugar
binding site.
Despite the open nature of the MBP-C binding site, all
monosaccharides are bound in a single orientation relative to the
Ca site 2 ligands. Modeling shows that if any of the
sugars is rotated 180° relative to Ca
ligands,
steric clashes do not occur between sugar and protein, and the
(+)-syn-clinal hydroxyl groups can still make the same
coordination and hydrogen bonds. However, there is no evidence in the
electron density maps for a mixture of two orientations in any of the
complexes. A similarity among all bound sugars, which may account for
the selection of a preferred binding orientation is that the less polar
surface of each sugar faces Val
(B face of Man, GlcNAc,
and Gal; A face of Fuc) (Fig. 7Fig. 8Fig. 9).
While the structures of
MBP-C-monosaccharide complexes do not explain subtle differences in
binding affinity, the structures shed light on certain differences
observed in MBP-A. In MBP-A, the affinity for -Me-Fuc is 5-fold
higher than for
-Me-Fuc, and this difference in affinity can be
eliminated by mutations at position 189(17) . These
observations led to the suggestion that Fuc is oriented in MBP-A with
the anomeric oxygen near position 189, opposite to that originally
proposed based on the orientation of Man (12) but like that
seen in the MBP-C-Fuc complexes (Fig. 7). The present results
confirm that this binding orientation is possible, although a
definitive answer requires the determination of MBP-A-Fuc complex
structures.
Figure 10:
Second binding site seen at 1.3 M -Me-Man. Stereo view of electron density (contoured at 1.0
) superimposed on
-Me-Man bound to the second binding site
in MBP-C. Dashed lines denote atoms within hydrogen-bonding
distance of N
of Lys
(the ring oxygen
(O-5), 6-OH, and 2-OH of
-Me-Man). The geometry of the three
oxygen atoms indicates that N
can only form two
hydrogen bonds with these atoms: the angle formed between 2-OH,
N
, and 6-OH is 95°, while the angle formed between
O-5, N
, and either 2-OH or 6-OH is 55°. This
arrangement suggests that O-5 acts as an acceptor of a bifurcated
hydrogen bond with either 2-OH or 6-OH.
MBPs require a broad monosaccharide
specificity in order to recognize a variety of cell surfaces. The
structures described here, as well as the previous structure of an
MBP-Aoligosaccharide complex(12) , demonstrate that MBPs
meet this requirement by having a very open binding site that is
specific only for a minimal subset of functional groups on the ligand.
The site selects ligands containing vicinal equatorial hydroxyl groups
with the same stereochemistry as that of the 3- and 4-OH of Man,
resulting in the formation of two coordination bonds with
Ca
, four hydrogen bonds with Ca
ligands, and a single apolar van der Waals' contact. The
absence of interactions between other portions of the sugar and protein
permits binding to a variety of sugars, including Man, Fuc, and GlcNAc.
The presence of alternative binding orientations
for Man in two highly related proteins highlights the pitfalls of
homology modeling. In particular, the orientation of Fuc proposed for
MBP-A based on the Man complex structure (12) has
formed the basis for modeling of fucosylated ligand binding to the
selectins(11, 14, 15, 16, 19) .
The structures presented here show that slight changes in protein
structure can effect significant changes in the orientation of the
bound sugar.
The different orientations of Man bound to MBPs A and C
may have implications for detailed specificity differences between
these two proteins. A previous study has shown that MBP-C binds a
different set of oligosaccharides than MBP-A, even though the two
proteins bind a similar spectrum of monosaccharides (9) (Table 5). In particular, binding to neoglycolipid
blots suggests that MBP-C preferentially recognizes the internal
Man core structure common to N-linked
oligosaccharides (residues 3, 4, and 4` in ), while MBP-A prefers terminal Man, Fuc, and GlcNAc
residues.
The results in the present study indicate that direct binding of
MBP-C to the Man core would require interaction with Man
residues 4 and/or 4`, since the 3-OH group of Man residue 3 is substituted in a glycosidic linkage. Similarly, further
substitution of Man residue 4` at the 3-OH, as occurs in the
case of high-mannose oligosaccharides, would preclude binding of MBP-C
to this residue, although interactions with 2-substituted Man residues
further out on this branch would still be possible.
If the
orientation of the sugar at Ca site 2 is reversed
between MBPs A and C, the disposition of other sugars on an
oligosaccharide chain with respect to the protein would differ greatly
and give rise to different interactions. When a Man
oligosaccharide binds to MBP-A, the penultimate sugar of each
branch interacts with the protein through ordered water molecules,
forming at most one direct contact with the protein (a poorly ordered
lysine residue)(12) . Thus an extended site is formed using
water molecules, and in this case there is no site on the protein
specific for a second sugar residue. The energetics of these
water-mediated sites are unclear, however, since this oligosaccharide
binds to MBP-A with the same affinity as Man(8) . Nonetheless,
it is clear that certain oligosaccharide conformations can be
sterically excluded from binding, if the orientation of the sugar at
the primary binding site is reversed between MBPs A and C.
In addition to the second
site observed here, it is possible that there is yet another
monosaccharide binding site in MBP-C, which is blocked by a lattice
contact in our crystal form. This would be surprising, however, because
sub-MBP-C crystals remain intact, retain the same unit cell dimensions,
and diffract to 1.8 Å after being soaked in 1.3 M -Me-Man for several hours. If a second monosaccharide-binding
site were blocked by a lattice contact, the crystal might be expected
to crack or at least change cell dimensions when soaked in a high
concentration of Man. As noted above, however, a second binding site
specific for a ligand larger than a single monosaccharide may not be
detected by soaking monosaccharides into an intact crystal. Ultimately,
the determination of the structure of a complex between MBP-C and an
oligosaccharide will be needed to see whether oligosaccharide binding
requires an extended site or multiple sugar binding sites.
The relationship of protomers in the MBP-A dimer
differs significantly from the relationship of protomers in the trimer,
but the same residues of the CRD form the interfaces of both the dimer
and trimer(32) . In this regard, it is noteworthy that the
central contact in the MBP-C dimer interface, which occurs between
Leu and a pocket formed by the other protomer (Fig. 4), is very similar to the central contact of the
coiled-coil neck-CRD interface in the MBP-A trimer. In that case,
Phe
from the neck domain is buried in a pocket formed by
Thr
, Asn
, Leu
,
Leu
, and Cys
of the CRD of a different
protomer(32) . The residues that form this pocket are
equivalent to those that form the pocket in the MBP-C dimer interface
(Met
, Leu
, Leu
, and
Cys
), and Phe is present at position 106 of MBP-C,
corresponding to Phe
of MBP-A. Therefore, it is likely
that Phe
forms an integral part of the neck-CRD interface
of MBP-C, and that the neck-CRD interaction seen in the MBP-A trimer is
similar in MBP-C. On the other hand, the fact that the MBP-A and MBP-C
dimer structures are so different suggests that sequence differences in
the neck-CRD interface may also produce significant differences in the
relative disposition of the neck and CRD between the MBP-A and MBP-C
trimers. This would in turn create a different spacing of
carbohydrate-binding sites in the two proteins and give rise to
different avidites that depend on the spacing of monosaccharides in
multivalent ligands. Resolution of this important issue awaits
determination of a trimeric MBP-C structure.
The atomic coordinates (codes 1RDI through 1RDO, inclusive) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.