The mammalian hepatic asialoglycoprotein
receptor, a member of the C-type animal lectin family, displays
preferential binding to N-acetylgalactosamine compared with
galactose. The structural basis for selective binding to
N-acetylgalactosamine has been investigated. Regions of the
carbohydrate-recognition domain of the receptor believed to be
important in preferential binding to N-acetylgalactosamine
have been inserted into the homologous carbohydrate-recognition domain
of a mannose-binding protein mutant that was previously altered to bind
galactose. Introduction of a single histidine residue corresponding to
residue 256 of the hepatic asialoglycoprotein receptor was found to
cause a 14-fold increase in the relative affinity for
N-acetylgalactosamine compared with galactose. The relative
ability of various acyl derivatives of galactosamine to compete for
binding to this modified carbohydrate-recognition domain suggest that
it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with
N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the
N-acetyl substituent of the sugar. Evidence for the role of
the side chain at position 208 of the receptor in positioning this key
histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a
hydrogen bond to the imidazole side chain. When this serine residue is
changed to valine, loss in selectivity for
N-acetylgalactosamine is observed. The structure of this
mutant reveals that the
-branched valine side chain interacts
directly with the histidine side chain, resulting in an altered
imidazole ring orientation.
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INTRODUCTION |
The mammalian hepatic asialoglycoprotein receptor is best known
for its role in clearance of desialylated proteins from serum (1, 2).
The receptor is often regarded as a galactose-binding animal lectin
because of its ability to bind galactose exposed when sialic acid is
removed from complex N-linked oligosaccharides. However, the
affinity of the receptor for
N-acetylgalactosamine-terminated sugar structures and
neoglycoproteins derivatized with this monosaccharide is substantially
higher than for those terminating in galactose (3, 4). In competition
assays, N-acetylgalactosamine
(GalNAc)1 competes
approximately 60-fold more effectively than galactose (Gal) for binding
to the carbohydrate-recognition domain (CRD) of the major subunit of
the receptor (rat hepatic lectin 1, RHL-1). Interestingly, preferential
binding of GalNAc is not a property of the asialoglycoprotein receptor
of peritoneal and tumoricidal macrophages, even though the CRD of this
receptor shares 85% sequence identity with RHL-1 (5, 6).
The basis for the different behavior of the hepatic and macrophage
asialoglycoprotein receptors has previously been investigated by
studying a series of chimeric CRDs containing different portions of the
two receptors (7). This approach led to identification of three regions
of the primary structure that contribute to selective binding of
GalNAc. The position of the most significant of these regions relative
to the monosaccharide binding site found in the three-dimensional
structure of the homologous mannose-binding protein (MBP) CRD (8)
suggests that these amino acids are unlikely to form direct contacts
with the bound sugar. Instead, these residues probably influence the
position of a critical histidine side chain (His256 in
RHL-1), which could in turn contact the N-acetyl portion of the sugar. The importance of His256 for GalNAc selectivity
was confirmed by mutagenesis (7).
Direct structural analysis of the CRD from an asialoglycoprotein
receptor has not yet proven feasible. However, studies on mutant forms
of the homologous mannose-binding CRD of rat serum mannose-binding
protein suggest the likely arrangement of Gal and GalNAc in the binding
site of the receptor. Insertion of three portions of RHL-1 into MBP
results in a complete change in ligand-binding preference of this CRD
from mannose to galactose (9). The mutant QPDWG, which contains the
sequence Gln-Pro-Asp-Asp-Trp-Tyr-Gly-His-Gly-Leu-Gly-Gly in place of
residues 185 through 191 of MBP, has binding properties almost
indistinguishable from the asialoglycoprotein receptor when galactose
and mannose are compared. Crystallographic analysis of galactose bound
to the mutant CRD provides a model for the mode of galactose binding to
the asialoglycoprotein receptor (10).
These studies have now been extended by further mutagenesis of the CRD
from MBP to mimic the selective binding of GalNAc by RHL-1. Structural
analysis confirms the importance of the histidine corresponding to
His256 of RHL-1 in directly contacting the bound ligand and
provides additional evidence that other regions of RHL-1 that
contribute to GalNAc binding may do so indirectly by influencing the
position of this key histidine side chain.
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EXPERIMENTAL PROCEDURES |
Preparation of Mutant Proteins--
Site-directed mutagenesis
was conducted using double-stranded synthetic oligonucleotides to
replace restriction fragments of the cDNA for MBP following
standard methods (11). Oligonucleotides were prepared on an Applied
Biosystems 391 DNA synthesizer. The CRDs derived from MBP were
expressed in the pINIIIompA vector as described previously (9) and
those derived from RHL-1 were expressed in vector pT5T (7). All of the
CRDs were isolated by chromatography on galactose-Sepharose (7,
12).
Sugar Synthesis--
N-propionyl and
N-iso-butanoyl derivatives of galactosamine were prepared by
reaction of the free amino sugar with propionic and
iso-butyric anhydride (13). Thio derivatives were prepared as described previously (14). The carboxypropionyl derivative was
synthesized by reacting galactosamine hydrochloride with succinic anhydride. The detailed procedure will be published elsewhere. All
derivatives were characterized using 1H-NMR spectroscopy
and fast atom bombardment mass spectroscopy. Concentrations were
determined using the Morgan-Elson assay (15) following hydrolysis in 1 M HCl for 3 h at 96 °C.
Binding Assays--
Solid phase binding and competition assays
were performed using 125I-Gal34-serum albumin
as reporter ligand (7). The KI values reported
represent the point of half-maximal competition for binding as
determined using a nonlinear least squares fitting program (SigmaPlot,
Jandel Scientific).
NMR Analysis--
Proteins for NMR studies were further purified
by reverse phase chromatography on a C3 ultrapore column (Beckman
Instruments) using a gradient from 10 to 50% acetonitrile in the
presence of 0.1% trifluoroacetic acid. Eluted proteins were
lyophilized and prepared for NMR analysis as in previous studies (16).
Sugar was added in aliquots from concentrated stock solutions in
D2O. Titrations were performed on a Varian Unity 500 spectrometer. Changes in chemical shift were fitted to a simple first
order binding equation, 
= 
bound/(KD + [sugar]), where KD is the dissociation constant and

bound is the apparent change in chemical shift for
the bound form.
Crystallization and Data Collection--
Protein prepared as
described above was subjected to clostripain (Worthington Enzymes)
digestion and repurified by affinity and reverse phase chromatography
as described previously (17). Lyophilized protein was redissolved in 10 mM NaCl and 10 mM CaCl2 to 10-20
mg/ml, and the pH was adjusted to 7 by addition of dilute sodium
hydroxide. Crystals of QPDWGH and QPDWGHV were grown at 20 °C by
hanging drop vapor diffusion by mixing equal volumes of protein with
reservoir solutions containing 12-15% polyethylene glycol 8000, 100 mM Tris-HCl, pH 8.0, 20 mM CaCl2,
10 mM NaCl, 0.02% NaN3. Crystals typically
grew in 5-7 days to a size of 0.3 × 0.3 × 0.2 mm. Prior to
data collection, the crystals were adapted in a stepwise manner to
reservoir solution containing 0, 5, 7.5, 10, 15, and 20%
2-methyl-2,4-pentanediol. The solutions all contained 200 mM GalNAc (Sigma) to form the monosaccharide complexes.
Crystals were flash-cooled at 100 K, and diffraction data were measured on a RAXIS II imaging plate detector mounted on a rotating copper anode. Data were processed using DENZO and SCALEPACK (18).
Structure Solution and Refinement--
Crystals of QPDWGH and
QPDWGHV are nearly isomorphous with those of the galactose-binding
MBP-A mutant QPDWG, with one trimer comprising the asymmetric unit
(10). Structure solution consisted of rigid body refinement of the
QPDWG mutant model against each of the data sets from QPDWGH and
QPDWGHV. Water molecules, Ca2+ ions, and the side chain of
residue 202 were omitted from the model for both data sets.
Additionally, the side chain of residue 154 was omitted during the
initial QPDWGHV refinement. Temperature factors from the QPDWG model
were retained. The protomers were refined as individual rigid bodies
against data from 10-4.0 Å and then from 10-2.8 Å. Positional and
temperature factor refinement followed using data from 10-2.2 Å.
Calcium atoms were added, and the omitted residues were built into the
difference (Fo-Fc) electron density using the program O (19). From this
point on, a maximum likelihood refinement target and all data from 30 Å to the high resolution limit were used (20). Water molecules were
added, and positional and isotropic temperature factor refinement was carried out. A bulk solvent correction and an overall anisotropic temperature factor tensor were applied throughout. The GalNAc molecule
was modeled only after the difference density allowed unambiguous
positioning of the sugar ring. All calculations were performed using
the program CNS (21). Data collection and final refinement statistics
are given in Tables I and
II.
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Table I
Data collection and refinement statistics, diffraction data
The space group is C2 with the trimer forming the asymmetric unit. The
numbers in parentheses correspond to values in the highest resolution
shell, which is 2.0-2.09 Å for QPDWGH and 2.1-2.2 Å for QPDWGHV.
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Table II
Data collection and refinement statistics, model geometry
All residues except Asn206 of one protomer in QPDWGH + GalNAc, which lies in a poorly ordered turn, fall within the allowed
regions of the Ramachandran plot. The GalNAc in protomer 1 of QPDWGH
was modeled as a mixture of both the and anomer with
occupancies of 0.65 and 0.35, respectively. A sodium ion was modeled in
the QPDWGH + GalNAc structure but was not seen in the QPDWGHV + GalNAc structure. No other significant differences are observed among
the different copies except in regions of lattice contacts. The side
chains of His99 and Met103 in protomer 1 were modeled
in two conformations in QPDWGHV + GalNAc.
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RESULTS |
GalNAc-specific Binding to Mutants of MBP--
As a complement to
previous studies of the hepatic and macrophage asialoglycoprotein
receptors (7), four regions of the hepatic receptor found to be
important for selective binding of GalNAc were introduced into the CRD
of MBP (Fig. 1). The starting point for
these studies was a mutant CRD (QPDWG) already containing three regions
from RHL-1, which are sufficient to establish high affinity binding to
galactose (9). Because the histidine at position 256 of RHL-1 is
absolutely essential for highly selective binding of GalNAc, this
residue (region 4) was introduced into the corresponding portion of
mutant QPDWG in place of Thr202 to create mutant QPDWGH.
The effect of this change was to increase by 14-fold the relative
ability of GalNAc to compete for binding compared with Gal (Table
III). This result confirms the importance of this histidine residue in binding to GalNAc.

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Fig. 1.
Sequence comparison of the CRDs of MBP and
RHL-1. A, amino acid sequences of the carbohydrate
recognition domains of MBP and RHL-1. The sequence of the C-terminal
portion of rat serum mannose-binding protein (MBP-A) is
shown along with changes that have previously been made to generate
high affinity binding to galactose. Elements of secondary structure in
the crystal structure of MBP are marked H, S, and
L for -helix, -strand, and loop. Essential residues in
regions 1-4 of RHL-1 that have been implicated in selective binding of
GalNAc are indicated. The residue numbering scheme for the QPDWGH
mutant is shown. B, ribbon drawing of QPWDG sugar binding
site showing the MBP residues equivalent to those in regions 1-4 of
RHL-1. The structure of the QPDWG mutant complexed with GalNAc is
depicted in ribbon form (10). GalNAc and amino acid chains are drawn as
balls-and-sticks, calcium ions are drawn as gray
spheres, and the glycine-rich loop is shaded gray. This
figure and Figs. 4 and 6 were prepared with MOLSCRIPT (23).
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Table III
Analysis of sugar binding to mutant forms of mannose-binding protein
All mutants contain the sequence
Gln185-Pro-Asp-Asp-Trp189 plus the inserted sequence
Gly-His-Gly-Leu-Gly-Gly in place of residue Ser191 to establish
high affinity galactose binding.
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The higher affinity of the MBP derivative containing His202
for GalNAc compared with Gal was independently verified using NMR to
assess binding affinities directly. The affinities were determined by
examining the perturbation of amino acid side chain resonances in the
aromatic region of the one-dimensional proton NMR spectrum of the
His202 mutant with increasing concentrations of sugar. The
measured KD of 2.1 mM for Gal was
comparable with that previously determined for the parental
Thr202 mutant (16). However, similar analysis of GalNAc
binding provided a KD value of 0.20 mM
(Fig. 2). Thus, the NMR titrations provide independent evidence that the His202 mutant
displays more than 10-fold higher affinity for GalNAc than for Gal.

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Fig. 2.
Binding of GalNAc to QPDWGH mutant of MBP
measured by NMR. Changes in the chemical shift of the resonance at
6.01 ppm in one-dimensional proton NMR spectrum ( ) of the mutant
protein were used to detect binding of sugar. The experimental points
are indicated as filled circles, and the fitted curve is
shown as a solid line.
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Examination of individual spectra obtained during the titration also
provides information about the nature of the interaction between
His202 and GalNAc. As shown in Fig.
3, a well resolved resonance can be
associated with H2 (H
1) of the imidazole ring of this residue by
comparison with the parental CRD. In the presence of increasing concentrations of sugar, the peak is largely unchanged, although there
may be some broadening associated with immobilization at higher
concentrations. However, it is difficult to quantify this effect
because of increasing interference from the adjacent resonance that
shifts upfield from 7.65 ppm with increasing sugar concentrations. Shifting of this adjacent resonance is also observed in both the parental and His202-containing CRDs titrated with
galactose.

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Fig. 3.
One-dimensional proton NMR spectrum of mutant
MBPs. The bottom trace shows a portion of the spectrum of the
galactose-binding QPDWG mutant of MBP. A single histidine-derived
resonance, corresponding to H2 of His116, is evident in
this portion of the spectrum (other histidine H2 resonances lie further
upfield) (14). The remaining traces show the spectrum of the
His202-containing mutant QPDWGH in the presence of
increasing concentrations (0-4 mM) of GalNAc. The new
resonance for H2 of His202 appears at 7.8 ppm.
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In cases where sugar protons are positioned above aromatic amino acid
side chains in sugar-lectin complexes, there is often a ring
current-induced broadening and shifting of the resonances (22). No such
effects are observed for the methyl protons in the acetamido
substituent of GalNAc in the presence of the
His202-containing CRD. This result suggests that there is
no direct stacking interaction with the face of the imidazole side
chain of His202. Titration of the CRD from RHL-1 similarly
fails to show broadening of this resonance (data not shown), providing
further evidence for the similarity of the interaction of RHL-1 and the
MBP mutant with GalNAc.
Crystallographic Analysis of a GalNAc-specific MBP Mutant--
The
crystal structure of QPDWGH complexed with GalNAc was determined. The
structure is essentially superimposable with the original QPDWG
structure. The Thr202
His mutation produces significant
changes only at the His202 and Ser154 side
chain positions (Fig. 4). The hydroxyl
group of Ser154 in QPDWGH swings about the
1
angle by 120° relative to its orientation in QPDWG and forms a
hydrogen bond with His202. The orientation of
His202 confirms that the increase in GalNAc specificity of
this mutant results from a direct interaction between this residue and
GalNAc. The imidazole ring is oriented so that the methyl group in the acetamido substituent of GalNAc makes van der Waals contact with N
2
and C
1 of His202 (Fig. 5).
The contact distances between the acetamido carbon atom and the N
2
and C
1 atoms are 3.9 ± 0.2 and 3.9 ± 0.1 Å, respectively (averaged over the three copies in the trimer), and compare favorably with the expected van der Waals distance between two
carbon atoms or between a carbon and a nitrogen atom.

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Fig. 4.
Ribbon representation of crystal structure of
the QPDWGH mutant of MBP complexed with GalNAc. Stereo ribbon
drawing shows the vicinity of the GalNAc binding site with the sugar
and selected residues drawn as balls-and-sticks. The
glycine-rich loop stacked against Trp189 is highlighted in
gray and Ca2+ 1 and 2 are shown as gray
spheres. The hydrogen bond between Ser154 and
His202 is drawn as a dashed line.
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Fig. 5.
van der Waals dot surface representation of
the GalNAc binding site of the QPDWGH mutant of MBP. Stereo pair
shows the GalNAc binding site in an orientation very similar to that in
Fig. 4. The His202/GalNAc contact is apparent at the
bottom. The stacking of the glycine-rich loop, Trp189 ring,
and the apolar face of GalNAc is seen at the top. This figure was
prepared with the Xfit component of the XtalView program suite
(24).
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Previous studies with a macrophage galactose receptor mutant that
exhibits more than 20-fold enhanced binding of GalNAc compared with Gal
have indicated that histidine at the position corresponding to
His256 of RHL-1 has a unique ability to support
preferential binding of GalNAc (7). This residue has been mutated to a
variety of different amino acids. Substitution of either alanine or
glutamine at this position reduces preferential binding of GalNAc over
Gal to less than 2-fold, whereas changes to asparagine, tyrosine, aspartic acid, and lysine actually result in lower affinity for GalNAc
than for Gal. Glutamine, which is similar in length to histidine and
contains amide and carbonyl oxygen functionalities, does not produce
strong GalNAc selectivity, suggesting that hydrogen bonding is not
involved and that the presence of C
1 is essential. Of the amino
acids tested, glutamic acid comes closest to histidine, because it
supports 4-fold tighter binding of GalNAc than Gal. The unique ability
of histidine to elicit strong GalNAc selectivity can be explained by
the fact that none of the other amino acids is isosteric with histidine
and thus cannot form the favorable van der Waals contacts with the
GalNAc acetamido group.
Role of Region 1 in GalNAc Selectivity--
Among the three other
regions of RHL-1 that affect GalNAc binding (7), region 1 shows the
largest effect but is somewhat unusual in that most amino acids at
position 208 of the hepatic receptor support selective binding to
GalNAc. The major exception is valine, which is found at the
corresponding position in the macrophage receptor. This substitution is
primarily responsible for the different binding properties of the
hepatic and macrophage receptors. The tolerance of this position for a
wide range of side chains other than valine suggests that the effect of
the valine might reflect alterations in the positions of other side chains, particularly His256 in region 4, caused by the
presence of a
-branched amino acid in region 1.
To provide evidence for this interpretation of the role of region 1, a
further mutation in RHL-1 was created by substitution of isoleucine at
position 208. This change results in substantial loss of affinity for
GalNAc, because the KI,
Gal/KI, GalNAc for this mutant is
1.9 ± 0.1. This value can be compared with 1.2 ± 0.1 in the
presence of Val208 and 60 ± 8 in the presence of
Asn208 (7). Combined with previous studies introducing
various amino acid substitutions into region 1 of the macrophage
galactose receptor, this result provides support for the suggestion
that the presence of a
-branched amino acid at the position of
Asn208 in RHL-1 leads to loss of affinity for GalNAc.
Threonine also has a
-substituent, but unlike valine and isoleucine,
it has a hydroxyl group that could hydrogen bond to His202
in a manner similar to that seen for Ser154 in QPDWGH.
The possibility that the corresponding portion of MBP might similarly
influence the binding of GalNAc to QPDWGH, perhaps by affecting the
position of the histidine side chain, was investigated by insertion of
valine in place of the residue that most nearly corresponds to
Asn208 of RHL-1. The alignment of amino acid sequences
shown in Fig. 1 suggests that there are some differences in the
conformation of this region of the protein, because the segment
connecting
-helix 2 and
-strand 2 in MBP is 1 residue shorter
than the corresponding segment of RHL-1. However, Ser154 of
MBP, at the beginning of
-strand 2, appears to correspond most
closely to Asn208 of RHL-1. Changing this residue to valine
to create mutant QPDWGHV results in a 3-fold loss of preferential
binding of GalNAc (Table III). This finding is consistent with the
suggestion that amino acid side chains in region 1 influence the
ability of the histidine residue in region 4 to mediate higher affinity
binding to GalNAc.
The crystal structure of the His202/Val154
mutant (QPDWGHV) was determined to probe the structural basis of the
effect of Val154 on GalNAc selectivity (Fig.
6A). In this mutant, the
-methyl substituent of Val154 makes van der Waals
contact with the C
, N
1, and C
1 atoms of the His202
imidazole ring, which rotates about its
2 torsion angle
by 25° to accommodate the Val154
-methyl group (Fig.
6B). Despite this rotation, the imidazole ring of
His202 is still able to make van der Waals contact with the
GalNAc acetamido group (lines in Fig. 6A). These findings
are consistent with the observation that although the specificity of
QPDWGHV for GalNAc is reduced 3-fold (Table III), the presence of
Val154 does not completely abolish GalNAc selectivity. The
basis of the reduced preference for GalNAc is unclear, because the van der Waals contact distances between GalNAc and His202 are
not significantly altered when Ser154 is mutated to valine.
Given the difference in the length of segments connecting
-helix 2 to
-strand 2 in MBP and RHL-1, it is possible that valine at
position 208 in RHL-1 clashes more severely with His256,
resulting in a larger displacement of the imidazole ring than that
observed in QPDWGHV.

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Fig. 6.
Ribbon representation of crystal structure of
the QPDWGHV mutant of MBP complexed with GalNAc. A,
stereo ribbon drawing shows the GalNAc binding site in the same
orientation as in Fig. 4. The effect of Val154 can been
seen to cause a rotation of His202 about the
2 torsion angle as compared with the QPDWGH mutant (Fig.
4). The van der Waals contact is depicted as a set of parallel lines.
B, closeup of Fig. 6A showing the
His202 side chain from QPDWGHV (in gray)
superimposed on His202 from the QPDWGH structure (in
white). The relative rotation of 25° between the imidazole
rings is apparent.
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Incorporation of two additional regions of RHL-1 into the macrophage
receptor increases the relative effectiveness of GalNAc as a competitor
for binding by roughly 3-fold (7). The introduction of regions 2 and 3 (Fig. 1) into MBP does not result in significantly increased
selectivity for GalNAc (Table III). In fact, some loss in selectivity
is observed in the presence of either a short or long version of region
3. This loss of affinity for GalNAc may reflect an indirect effect on
the essential His202 in region 4, which is immediately
adjacent in the amino acid sequence. The lack of positive effects from
inclusion of regions 2 and 3 suggests that these portions of the MBP
mutants do not assume conformations that accurately mirror the
corresponding segment of the RHL-1. Alternatively, they may have
indirect effects in RHL-1 that are mediated by residues not present in
MBP.
Interactions of the N-Acyl Substituent--
To compare the
selectivity of the binding site in authentic RHL-1 with QPDWGH in
region 4, the abilities of different N-acylated derivatives
of galactosamine to compete for binding were assessed. The results for
the two proteins are summarized in Table
IV. The data for thio derivatives of the
N-acetyl and N-propionyl substituents reflect the
same relative preferences for side chain size previously observed for a
series of simple N-acyl derivatives (7). Thus, the
derivative with the larger thioacetyl substituent competes for binding
to RHL-1 more effectively than does GalNAc and appears to bind at least
as well as N-propionyl galactosamine. However, the
thiopropionyl derivative, like the n-butanoyl derivative, binds less well than the N-propionyl derivative. These
results define an optimal size for the 2-substituent in line with
previous studies (7). The change of oxygen to sulfur in the acyl
substituent has approximately the same effect as addition of a carbon
atom. Steric effects could also explain why the still larger
carboxypropionyl derivative is the least effective inhibitor, although
it is possible that the negative charge adversely affects the
interaction of this ligand with the protein.
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Table IV
Binding of acylated derivatives of galactosamine to CRDs
The first three values in column 1 are recalculated from Ref. 7. Ac,
acetyl; Pr, propionyl; nBu, n-butanoyl; iBu,
iso-butanoyl; SAc, thioacetyl; SPr, thiopropionyl; CPr,
3-carboxy propionyl.
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When the same derivatives were tested with the His202
mutant of MBP, similar effects of size were observed. The thio
derivatives show enhanced binding to the mutant MBP that closely
parallels that observed for the natural RHL-1 binding site, whereas the carboxypropionyl derivative is again less effective. These comparisons provide evidence that the mutant MBP displays a restricted binding site
for the 2-acylamido substituent of the ligand. The properties of this
binding site are therefore similar to, although not exactly like, those
of RHL-1. The apparently higher affinity of the propionyl derivative
for RHL-1 is not observed in the modified MBP, indicating that the
favorable interactions resulting from the presence of this
intermediate-sized side chain must involve regions of RHL-1 that have
not been incorporated into the MBP framework. However, the
N-iso-butanoyl derivative is considerably less effective
than the N-propionyl derivative as a competitor for binding
to both QPDWGH and RHL-1. These results suggest that the larger group clashes with portions of QPDWGH, thereby limiting the size of the
substituent that can be tolerated in a manner similar to RHL-1. Modeling suggests that although the N-propionyl derivative
can be accommodated in the QPDWGH sugar- binding site, one of the terminal methyl groups of the larger N-iso-butanoyl
derivative would clash with His202 and Asn210.
It is possible to model a N-iso-butanoyl rotamer such that
there are no steric clashes, but there would be an entropic cost to restricting the rotation of the butanoyl group. In the propionyl derivative, this methyl group would be absent and no unfavorable contacts would be expected, which is consistent with the binding data.
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DISCUSSION |
Several lines of evidence suggest that the modified GalNAc-binding
CRD in mutant QPDWGH provides a useful model for the structure of the
GalNAc-selective binding site in RHL-1. The preferential binding of
GalNAc compared with Gal and the relative selectivity of the model CRD
for different N-acyl derivatives parallels qualitatively if
not quantitatively the natural GalNAc-binding CRDs. In addition, the
absolute dependence on a histidine residue in region 4 combined with
the absence of evidence of an aromatic stacking interaction with the
N-acetyl group of GalNAc indicate that the molecular interactions which stabilize GalNAc in the binding site are similar in
the two cases.
The nature of this interaction is evident from the crystallographic
analysis of the QPDWGH mutant. The increase in GalNAc selectivity in
the mutant is the result of a direct van der Waals contact between the
critical histidine and GalNAc. Furthermore, a nearby valine in the
QPDWGHV mutant has been shown to influence this interaction because its
-methyl substituent directly alters the orientation of the histidine
imidazole ring. Taken together, these results explain the ability of
RHL-1 to bind GalNAc preferentially over Gal because of the presence of
a histidine residue at position 256 and the fact that
Asn208 lacks a
substituent. In the macrophage galactose
receptor, which also contains the critical histidine residue, a valine
residue is present at the position corresponding to Asn208
in RHL-1. The
-methyl group of this valine residue likely perturbs the ring orientation of the critical histidine residue and its van der
Waals contact to GalNAc, leading to a substantial loss of GalNAc
selectivity. However, the QPDWGHV mutant data cannot completely explain
the loss of GalNAc selectivity in the macrophage receptor because,
unlike the receptor, the mutant still exhibits 3-fold selectivity for
GalNAc over Gal (Table III). The structure of this region of the
protein may differ between MBP and RHL-1, or other regions of the
receptor that have not been incorporated into the QPDWGHV mutant
are required to eliminate GalNAc selectivity completely.
The preferential binding of acyl derivatives of certain sizes suggests
the presence of a binding cleft that accommodates the 2-substituent of
the ligand; greater stability is achieved up to a certain size,
probably because of additional interactions between protein and ligand,
but still larger side chains result in steric clashes that reduce
affinity. The binding and NMR data provide some insight into the nature
of the interactions that define this site. The fact that the thioacetyl
derivative binds with higher affinity than the acetyl derivative argues
against the presence of a hydrogen bond to the carbonyl oxygen in the GalNAc complex, because sulfur would be expected to be a weaker hydrogen bond acceptor than oxygen in this position. Combined with the
NMR and crystallographic evidence that the binding does not involve
packing of the methyl portion of the acetyl group against the face of
the histidine or any other aromatic residue, these data indicate that
the predominant interactions defining the binding cleft for the
2-substituent are simple van der Waals contacts with the edge of a
histidine ring.
We thank Dawn Torgersen for assistance in
preparation and analysis of some of the proteins and Maureen Taylor and
Ken Ng for useful comments on the manuscript.