From the Structural Biology Section, Laboratory of
Immunogenetics, NIAID, National Institutes of Health, Rockville,
Maryland 20852, the § Biochemistry, Cellular, and Molecular
Biology Program, Johns Hopkins University, School of Medicine,
Baltimore, Maryland 21205, and the ¶ Laboratory of Cellular and
Clinical Immunology, INSERM Unit 255, Curie Institute, 75005 Paris,
France
Received for publication, January 16, 2001
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ABSTRACT |
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Fc Fc receptors, which are expressed on the majority of hematopoietic
cells, play important roles in antibody-mediated immune responses. The
binding of antigen-bound immunoglobulins (Ig) to Fc receptors activates
their effector functions and leads to phagocytosis, endocytosis of
IgG-opsonized particles, as well as antibody-dependent cellular cytotoxicity. The three major types of Fc receptors are Fc Human Fc The Fc binding region on Fc The recent crystal structures of Fc Protein Expression, Purification, and Crystallization--
The
extracellular part of the human Fc
The complex of Fc and Fc Structure Determination--
After briefly soaking in
precipitant solutions containing 25% glycerol, crystals were flash
frozen at 100 K. X-ray diffraction data from single crystals of both
crystal forms were collected using an ADSC Quantum IV charge-coupled
device detector at the X9B beam line of the National Synchrotron
Light Source at the Brookhaven National Laboratory and processed with
HKL2000 (25). The hexagonal crystals belong to space group
P6522 with cell dimensions a = b = 114.9 and c = 301.4 Å and diffract
to 3.5 Å. Due to the large unit cell dimension along c in
the hexagonal crystals, data were collected at a 300-mm crystal to
detector distance with a small oscillation angle of 0.2°. The
orthorhombic crystals of space group
P212121 with cell
dimensions of a = 76.4, b = 102.8, and
c = 123.3 Å diffracted to 3.0 Å. Both crystal forms
contain one molecule of Fc
The structure of the Fc Overall Structure of the Complex--
Crystals of a human
Fc The Structure of Fc The Structure of Fc--
The Fc fragment of an IgG1 antibody
comprises two identical chains (A and B), and each consists of two
C1-type immunoglobulin domains, CH2 and CH3.
The overall shape of the Fc fragment resembles that of a horseshoe with
the two CH3 domains packing tightly against each other at
the bottom of the horseshoe and the CH2 domains held apart
by carbohydrate moieties attached to the glycosylation site
Asn297 from both chains forming the opening of the
horseshoe. Well defined electron density throughout the Fc allowed for
unambiguous tracing of residues Leu234 to
Ser444 in chain A and Pro232 to
Leu443 in chain B of Fc, including the lower hinge regions,
Leu234-Pro238. The structure of the Fc
fragment in complex with Fc The Interface between Fc
The receptor·Fc complex buries ~1453 Å2 of
solvent-accessible area (Fig.
3A). The interface between
Fc
A combination of salt bridges, hydrogen bonds, and hydrophobic
interactions contributes to the receptor·Fc recognition.
Specifically, the interface between Fc Comparison of the Structures of Receptor·Fc
Complexes--
Including the two crystal forms described in this work,
there are a total of four Fc receptor and Fc complex structures
available to date. A comparison among these structures reveals the
conformation flexibility of this receptor·ligand complex and helps to
explain the molecular interactions that differentiate the high from the low affinity receptors.
The two crystal forms of Fc
The comparison between the structure of the Fc
Detailed structural analysis shows that the interface area buried in
the high affinity Fc
Although the overall pattern of the receptor·Fc interactions, namely
a preference for hydrogen bonding in the Fc A-chain part of the
interface and hydrophobic contacts in the Fc B-chain part of the
interface are preserved in both the Fc
Our results suggest that multiple interactions contribute to the
observed receptor-ligand affinity difference and that the higher
affinity recognition includes more extensive hydrophobic interface area
as well as more prominent electrostatic interactions.
Conserved Receptor·Fc Binding Interface--
Of the 13 receptor
interface residues, four (Trp90, Trp113,
Lys131, and Gly159) are invariant among all
human Fc Fc Receptor IgG Subtype Specificities--
Fc The Contribution of Carbohydrate to the Fc
The structure of the receptor·Fc complex reveals potential roles for
carbohydrate in receptor·Fc recognition. The first is the potential
role of glycosylation at Asn297 in supporting the
structural framework of the Fc. The Fc fragment used in this work was
generated from a human IgG1 and is therefore glycosylated. Multiple
carbohydrate moieties were visible in the electron density extending
from Asn297 of both chains of Fc toward each other into the
inter-chain region, referred to as the carbohydrate core region.
Asn297 is located next to the receptor binding interface.
The carbohydrate moieties, however, are orientated away from the
interface making no specific contacts with the receptor. The
glycosylation is thus unlikely to influence the receptor·Fc interface
directly. However, the unique arrangement between the oligosaccharide
moieties and the polypeptide chains of Fc makes it possible for the
carbohydrate to affect the conformational stability of the receptor
binding epitopes (40). Specifically, the spacing and the orientation between the two CH2 domains may be influenced by the
presence of sugar attachments (Fig. 1A). Because the binding
of the receptor to Fc requires a particular orientation of the epitopes
on both chains of Fc, it makes the receptor·Fc interface sensitive to the relative position and orientation of the two CH2 domains.
A Model for Fc
Although the current structure offers an insight to antibody·Fc Comparison of Fc
The recognition mode of binding to the lower hinge of Fc may evolve
from the unique requirement of Fc Receptor-IgG Recognition and Autoimmune Diseases--
In addition
to their normal cellular functions in host immunity, Fc receptors mediate
antibody-dependent inflammatory responses and cytotoxicity
as well as certain autoimmune dysfunctions. Here we report the crystal
structure of a human Fc receptor (Fc
RIIIB) in complex with an Fc
fragment of human IgG1 determined from orthorhombic and hexagonal
crystal forms at 3.0- and 3.5-Å resolution, respectively. The refined
structures from the two crystal forms are nearly identical with no
significant discrepancies between the coordinates. Regions of the
C-terminal domain of Fc
RIII, including the BC, C'E, FG loops,
and the C'
-strand, bind asymmetrically to the lower hinge region,
residues Leu234-Pro238, of both Fc chains
creating a 1:1 receptor-ligand stoichiometry. Minor conformational
changes are observed in both the receptor and Fc upon complex
formation. Hydrophobic residues, hydrogen bonds, and salt bridges are
distributed throughout the receptor·Fc interface. Sequence
comparisons of the receptor-ligand interface residues suggest a
conserved binding mode common to all members of immunoglobulin-like Fc
receptors. Structural comparison between Fc
RIII·Fc and
Fc
RI·Fc complexes highlights the differences in ligand
recognition between the high and low affinity receptors. Although not
in direct contact with the receptor, the carbohydrate attached to the
conserved glycosylation residue Asn297 on Fc may stabilize
the conformation of the receptor-binding epitope on Fc. An
antibody-Fc
RIII model suggests two possible ligand-induced receptor aggregations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, Fc
, and neonatal Fc receptors. Except for the neonatal Fc receptor and Fc
RII (CD23), which are related structurally to class I
major histocompatibility antigens and C-type lectins, respectively, all
other known Fc receptors are members of the immunoglobulin superfamily
(1, 2). Among them, Fc
RI and Fc
RI1 are high affinity Fc
receptors for IgG and IgE, respectively, with dissociation constants
ranging from 10
8 to 10
10 M. All
other receptors for IgG, such as Fc
RII and Fc
RIII, are low
affinity receptors with dissociation constants ranging from 10
5 to 10
7 M (3-5). In
addition to variations in affinity, each receptor displays distinct IgG
subtype specificities. Unlike the high affinity receptors that can bind
monomeric antibodies, the low affinity receptors preferentially bind to
and are activated by immune complexes.
RIII exists as two isoforms, Fc
RIIIA and Fc
RIIIB, that
share 96% sequence identity in their extracellular
immunoglobulin-binding regions. Fc
RIIIA is expressed on macrophages,
mast cells, and natural killer cells as a transmembrane receptor. In
contrast, Fc
RIIIB, present exclusively on neutrophils, is anchored
by a glycosyl-phosphatidylinositol linker to the plasma membrane.
Although Fc
RIIIA associates with the immunoreceptor tyrosine-based
activation motif containing Fc
RI
-chain or the T cell receptor
-chain for its signaling, Fc
RIIIB lacks a signaling component.
Nevertheless, it plays an active role in triggering Ca2+
mobilization and in neutrophil degranulation (6, 7). In addition,
Fc
RIIIB, in conjunction with Fc
RIIA, activates phagocytosis, degranulation, and the oxidative burst that leads to the clearance of
opsonized pathogens by neutrophils. A soluble form of Fc
RIIIB was
reported to activate the CR3 complement receptor-dependent inflammatory process (8).
RII and Fc
RIII has been identified
through the work of chimeric receptors with Fc
RI as primarily the
membrane proximal domain, including both the BC and FG loops. Further
site-directed mutations have revealed several residues of the receptor
critical to Fc binding (9-11). Similar regions on the
-chain of
Fc
RI were also identified to be critical for IgE binding affinity
(12). The receptor binding site on Fc has been located through the
construction of chimeric IgG molecules and mutational analysis at the
lower hinge region, residues located in the hinge region between the
CH1 and CH2 domains and immediately adjacent to
the N terminus of the CH2 domain of IgG (13-15). In particular, residues 234-238 (Leu-Leu-Gly-Gly-Pro) of the lower hinge
of IgG1 have been implicated in the receptor binding. The corresponding
region of IgE has also been implicated in the Fc
RI binding (16).
Apart from the lower hinge region, a few residues on the
CH2 domain of an IgG2b were also suggested to interact with
the receptor (17). However, with the exception of the neonatal Fc
receptor, the molecular recognition between the Fc receptors and Fc
remains to be elucidated (18).
RI
, Fc
RIIA, and Fc
RIIB
have each revealed a conserved Ig-like structure, with particularly the
small hinge angle between the two Ig-like domains, which is unique to
the Fc receptors (19-21). We report here the crystal structure of a
human Fc
RIII in complex with the Fc portion of a human IgG1
determined from two crystal forms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RIIIb receptor, residues 1-172,
was expressed as Escherichia coli inclusion bodies and then
reconstituted in vitro as described previously (22). Fc
fragments of human IgG1 antibody were prepared by the papain digestion as previously reported (23, 24).
RIII was prepared by mixing both components
in a 1:1 molar ratio and concentrating to 8-15 mg/ml for
crystallization. Single crystals of orthorhombic and hexagonal forms
were obtained by vapor diffusion in hanging drops at room temperature
under slightly different crystallization conditions. Rod-shaped
crystals of the orthorhombic form were grown from 10% polyethylene
glycol 4000 and 50 mM Hepes at pH 6.5. They appeared after
2-3 days and grew to an average size of 0.05 × 0.05 × 0.2 mm in ~2 weeks. Crystals of the second form, hexagonal bipyramids, were crystallized from 5% polyethylene glycol 6000 and 50 mM Hepes at pH 6.0. Crystals were first observed after 4-5
days, and reached a maximum size 0.15 × 0.15 × 0.4 mm after
1 month.
RIII and one molecule of Fc in the
asymmetric unit.
RIII·Fc complex in both crystal forms was
determined by molecular replacement. Polyalanine models of Fc (PDB
accession number 1FC1) and Fc
RIII were used in rotation and
translation searches using 15-4 Å data for both crystal forms.
An I/
(I) cutoff of 2.0 was used for hexagonal
data during search procedures. The position of the Fc molecule was
determined in both crystal forms using AmoRe (26). Rotation and
translation searches using data from the orthorhombic crystal resulted
in an unambiguous solution with a correlation coefficient (CC) of 39%
and an R-factor (RF) of 51% (CC = 51% and RF = 49% after rigid-body refinement in
AmoRe). Molecular replacement searches using the hexagonal crystal data
yielded a solution for the Fc from the third highest rotation solution
that became the highest ranking translation solution with CC = 38% and RF = 52% (CC = 46% and
RF = 50% after rigid-body refinement in AmoRe). The
position of Fc
RIII was determined in both crystal forms using the
program EPMR (27) with a polyalanine model of Fc
RIII and the
position of the Fc molecule fixed. Clear solutions were obtained for
both crystal forms with CC = 56% and RF = 48%
for orthorhombic crystal and CC = 55% and RF = 48% for hexagonal crystal, respectively. After rigid-body refinement of individual domains of the Fc
RIII·Fc complex modeled as
polyalanine using CNS (28) most side chains had clear electron density
into which side chains were built in. Disordered side chains lacking electron density were built with occupancies set to zero. The positional and grouped B-factor refinement was carried out
using maximum likelihood as a target function with CNS version 0.9. Model adjustments and rebuilding were done using the program O (29).
Carbohydrate molecules were added manually using 2Fo
Fc electron density maps contoured at 1.0
and
refined. The final model includes residues 5-172 of Fc
RIII,
residues 235-444 for one chain of Fc, and residues 233-443 for the
other chain of Fc.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RIII receptor in complex with a human Fc fragment of IgG1 were
grown in two forms under different conditions. The orthorhombic
crystals belong to the space group of
P212121 and diffract to
3.0-Å resolution, whereas the hexagonal crystals have
P6522 space group symmetry and diffract to
3.5-Å resolution. The structure of the complex was determined by
molecular replacement in both forms and refined to their resolution
limit. The final R-factors are Rcryst = 23.0% and Rfree = 28.9% for the orthorhombic form and Rcryst = 24.9% and
Rfree = 32.6% for the hexagonal form, respectively (Table I). The electron
density is continuous throughout the complex in the final
2Fo
Fc map except for three
surface loops of Fc
RIII (residues 31-34, 99-105, and 142-149)
located opposite from the Fc interface region. Despite different
crystal packing and solvent contents (57% in the orthorhombic and 64%
in the hexagonal form), both crystals contain one Fc
RIII and one Fc
molecule in each asymmetric unit, suggesting a 1:1 stoichiometry for
the binding between the receptor and Fc (Fig. 1). This is consistent with earlier
binding studies using non-equilibrium and equilibrium gel filtration
experiments (22). The conformation of the Fc
RIII·Fc complex is
essentially identical in both crystal forms, including the conformation
of the visible carbohydrate moieties on the Fc fragment (Fig.
2A).
Data collection and refinement statistics
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Fig. 1.
Ribbon drawing showing front view
(A) and side view (B) of the
Fc RIII·Fc. The side view of the
complex is rotated
90° from the front view. Fc
RIII is shown in
green, Fc is cyan. Positions of D1 and D2 domains
of Fc
RIII as well as CH2 and CH3 domains of
Fc are marked. Carbohydrate moieties are shown in
gray.
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Fig. 2.
A, superposition of an -carbon trace
of the Fc
RIII·Fc complex determined from both the orthorhombic
(green and cyan for Fc
RIII and Fc,
respectively) and hexagonal (orange and red)
crystal forms. B, superposition of the structure of
Fc
RIII in the receptor·Fc complex (green) with that of
ligand free receptor (orange). C, superposition
of the structure of Fc in Fc
RIII·Fc complex (cyan) with
that of unbound Fc (red).
RIII--
The structure of Fc
RIII in both
the orthorhombic and the hexagonal crystal forms can be readily
superimposed with the structure of ligand free receptor resulting in
r.m.s. differences between the individual domains of 0.6-0.8 Å among
all C
atoms (Fig. 2B) (22). The hinge angle between the
N-terminal (D1) and the C-terminal (D2) domains is 60°, which is
slightly larger than the 50° value observed in the ligand free
receptor. However, no significant change in the receptor conformation
is observed upon complex formation (Fig. 2B).
RIII does not differ significantly from
that observed in the structures of an unbound Fc fragment and a murine
intact IgG2a antibody (30, 31) (Fig. 2C). However, the
2-fold symmetry relating the two chains of Fc in other unligated Fc
structures, is no longer retained in the structure of the complex. The
horseshoe-shaped Fc is slightly more open at the N-terminal end of the
CH2 domains in the Fc
RIII·Fc structure compared with
other known structures of Fc. The hinge angle between CH2
and CH3 domains of chain A (Fc-A) is 95° and 100° in
the orthorhombic and the hexagonal crystals, respectively, ~10°
larger than the corresponding angle of chain B (Fc-B) and the
84°-89° angle observed in all structures of ligand-free Fc (Fig.
2C).
RIII and Fc--
The receptor binds to
Fc at the center of the horseshoe opening making contacts to the lower
hinge regions of both A and B chains of Fc (designated here as Hinge-A
and Hinge-B, respectively, for the lower hinges of Fc-A and Fc-B) (Fig.
1). Such binding breaks down the dyad symmetry of the Fc, creating an
asymmetric interface whereby the identical residues from Hinge-A and
Hinge-B interact with different, unrelated surfaces of the receptor.
Furthermore, it excludes the possibility of having a second receptor
interacting with the same Fc molecule, resulting in a 1:1 stoichiometry
for the receptor·Fc recognition. The structural implications of the activation of Fc receptors is profound. Particularly, the 1:1 receptor·Fc binding stoichiometry highlights the importance of antigen in the receptor aggregation. In contrast to the high affinity Fc
RI and Fc
RI receptors, the binding of immunoglobulins to
Fc
RIII in the absence of antigen does not lead to receptor
aggregation. It can be argued that a 1:1 receptor-ligand stoichiometry
ensures the need for antigens in forming the receptor aggregation by
eliminating the possibility of Fc-mediated receptor aggregation as
suggested in a 2:1 stoichiometry. Precluding receptor aggregation
mediated by Fc alone also eliminates the potential deleterious effect
of antibodies whose concentration in vivo are often much
higher than that of antigen.
RIII and Fc molecules shows poor shape complementarity with a mean
shape correlation statistic of 0.53 (32), less than those between
T-cell antigen receptor and Class I major histocompatibility complex
molecules, between adhesion receptor CD2 and CD58, and between antibody
and antigen complexes. On the receptor side, all the contacts to Fc are
made exclusively through its D2 domain. The receptor D1 domain is
positioned above the Fc-B and makes no contacts with Fc (Fig. 1A). The interface of the complex consists of the hinge loop
between the D1 and D2 domain of the receptor, the BC, C'E, and FG
loops, and the C'
-strand. The BC loop is positioned across the
horseshoe opening making contact with residues of both Hinge-A and
Hinge-B. The C'-strand is situated atop the Fc-A leading to the C'E
loop in contact with residues of Hinge-A. The FG loop of Fc
RIII
protrudes into the opening between the two chains of Fc (Fig.
1A). All three receptor loops (BC, C'E, and FG) were
implicated in Fc binding through earlier studies of chimeric
Fc
RII/Fc
RI receptors and through site-directed mutagenesis (9,
10, 12). On the Fc side of the complex, interactions with the receptor
are dominated by residues Leu234-Pro238 of the
lower hinge (Table II), consistent with
results form earlier mutational studies (2). In particular, Hinge-A and
-B together contribute ~60% of the overall receptor·Fc interface
area (Fig. 3A). Interestingly, both Hinge-A and -B are found
disordered in all known Fc structures to date, including the structure
of an intact mouse IgG2a (30, 33-35). In contrast, residues of both Hinge-A and -B are clearly visible in the electron density maps from
both crystal forms, suggesting that the binding of Fc
RIII stabilizes
the lower hinge conformation of Fc.
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Fig. 3.
Fc RIII·Fc
interface. A, surface representation of Fc
RIII. The
interface region is color-coded. Regions involved in
the interactions with Hinge-A and Hinge-B are yellow and
green, respectively. All other contact areas are colored in
blue. Lower hinge regions of Fc are in
ball-and-stick representation (red).
B, interactions between Fc
RIII (green) and
chain A of Fc (cyan). The glycosylation residue,
Asn297, is also shown. C, interactions between
Fc
RIII (green) and chain B of Fc (cyan). The
side chain of Val158 is omitted for clarity. There is a
hydrogen bond between carbonyl group of Val158 and backbone
nitrogen of Gly236 (Table II) that is not shown in the
picture. Residues are colored by molecule. Important
hydrogen bonds are represented by dotted lines. The BC, C'E,
and FG loops as well as the C and C'
-strands of Fc
RIII, which
play an important role in the interactions, are labeled. Some secondary
structure elements of Fc lying behind and not contributing to the
binding shown as semi-transparent. Carbohydrate moieties
have been omitted for clarity.
Interface contacts between FcRIII and Fc
RIII and Fc-A is dominated by
hydrogen bonding interactions, whereas the hydrophobic interactions
occur primarily at the interface between Fc
RIII and Fc-B. There are a total of nine hydrogen bonds between the receptor and Fc, forming an
extensive network involving both the main-chain and side-chain hydrogen
bonding interactions (Fig. 3, B and C, and Table
II). Seven hydrogen bonds are distributed across the receptor and Fc-A interface and two are at the receptor and Fc-B interface. Alanine mutations, such as the H134A mutant of Fc
RII that resulted in the
loss of two interface hydrogen bonds, have been shown to reduce the
receptor·Fc binding drastically, illustrating the importance of the
interface hydrogen bonding network to the stability of the complex
(10). A hydrophobic core is formed between Trp90,
Trp113 of the receptor, and Pro329 of the
CH2 domain of Fc-B (Fig. 3C). This hydrophobic
core extends further to include Val158, the aliphatic side
chain of Lys161 of the receptor and Leu235 of
Hinge-B. Mutations of both Trp113 and Lys161 in
Fc
RIII lead to the loss in receptor function (11, 36). The side
chain of Leu235 on the Fc-B packs tightly against
Gly159 of the receptor leaving little space to accommodate
any residues larger than Gly at this position. A G159A mutation on
chimeric Fc
RII resulted in the complete disruption of Fc binding,
presumably due to the steric hindrance between Leu235 and
the
-carbon of the alanine mutant at position 159 (9). Of particular
interest is Trp113 of the receptor, which when mutated to
Phe resulted in the loss of Fc binding. This residue is not only part
of the interface hydrophobic core but also functions as a wedge
inserted into the D1 domain to stabilize the acute inter-domain hinge
angle between D1 and D2 domains of Fc
RIII. A W113F mutation would
result in the loss of this wedge and lead to a disruption in binding by altering the orientation between the D1 and D2 domains.
RIII·Fc complexes determined in the
present study are essentially identical and can be readily superimposed
with a root mean square (r.m.s.) deviations of 1.1 Å among all C
atoms. The superposition of the hexagonal form onto the published
Fc
RIII·Fc complex resulted in an r.m.s deviation of 0.5 Å for all
C
atoms (37) (Fig. 4A). An
analysis of the interdomain hinge angles shows that the
CH2-CH3 hinge angle is 10° larger in the
structure of the Fc
RIII·Fc complex than it is in the structure of
an intact IgG2a antibody (35) or the structures of ligand-free Fc (30)
(Table III). This results in a slightly
more open conformation of the Fc when ligated to the receptor. Apart
from the small change of the hinge angle, neither the Fc nor the
receptor displays significant conformational change upon complex
formation. The agreement between the orthorhombic and hexagonal crystal
forms of the complex indicates a well defined receptor-ligand
recognition free from conformational flexibility.
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Fig. 4.
Superposition of the
Fc RIII·Fc complex determined from the
orthorhombic crystal form onto the structures of previously determined
complexes Fc
RIII·Fc (46) and
Fc
RI·Fc (45). A,
superposition of the Fc
RIII·Fc complex determined from the
orthorhombic crystal form (green and cyan for
Fc
RIII and Fc, respectively) and previously determined structure of
Fc
RIII·Fc complex (46) (orange and red for
Fc
RIII and Fc, respectively). B, superposition of the
structure of Fc
RIII·Fc (green and cyan for
Fc
RIII and Fc, respectively) and Fc
RI·Fc (orange and
red for Fc
RI and Fc, respectively) complex (45).
C, a definition of the hinge angles. D, an
enlarged view of superposition of Fc
RIII·Fc (green and
cyan) and Fc
RI·Fc (red and
orange) complexes in the interface area. The lower hinge
regions are labeled. The view is identical to that in
B.
Hinge angle comparison of free and receptor complexed Fc
RIII·Fc complex and
that of the Fc
RI·Fc complex has provided further insight into the
molecular basis of the receptor affinity (38). Overall, a similar mode
of receptor-ligand recognition was observed in both the Fc
RIII·Fc
and the Fc
RI·Fc complexes with an r.m.s. deviation of 1.5 Å between all the C
atoms. In fact, most of the structural difference
resulted from the small variation between the
CH2-CH3 and C
3-C
4 interdomain hinge
angles (Fig. 4, B and C). This angle is ~10°
smaller in the Fc
RI·Fc complex structure, resulting in a slightly
closed conformation of Fc compared with that of the Fc
RIII·Fc complex.
RI·Fc complex (1850 Å2) is 400 Å2 more than that in the low affinity Fc
RIII·Fc
complex (1453 Å2). This is primarily due to a more
extensive interaction observed between the receptor and the non-lower
hinge residues of Fc in the high affinity complex than in the low
affinity receptor complex. Of the total interface area of the Fc, the
lower hinge and non-lower hinge regions contribute 870 and 580 Å2, respectively, in the Fc
RIII·Fc structure. The
corresponding regions contribute 740 and 1110 Å2,
respectively, in the Fc
RI·Fc structure. This results in
approximately twice as much interface area contributed by non-lower
hinge residues in the high affinity receptor·ligand complex than in
the low affinity receptor·ligand complex. Structurally, the lower
hinge of IgE-Fc adopts a very different conformation than that
of IgG-Fc in their respective receptor complexes (Fig. 4D).
This conformation difference may enable the high affinity Fc
RI to
interact more extensively with its ligand.
RIII·Fc and Fc
RI·Fc complexes, significant differences were also observed. First, there are more extensive hydrophobic interactions between Fc
RI and
IgE-Fc than those between Fc
RIII and IgG-Fc. Although the tryptophan-proline sandwich formed by Trp90,
Trp113 of Fc
RIII and Pro329 of Fc-B (the
corresponding Trp87, Trp110, and
Pro426 residues in Fc
RI·Fc) is preserved in both
structures, additional bulky residues, such as Trp130,
found in both Fc
RI and IgE-Fc may also contribute to stronger hydrophobic interactions in the Fc
RI·Fc complex compared with that
of the Fc
RIII·Fc complex. Second, a more extensive network of
hydrogen bonds and salt bridges exists at the Fc
RI·Fc interface compared with that of Fc
RIII·Fc. Furthermore, the hydrogen bonds in the Fc
RI·Fc interface are formed mostly between the side-chain atoms whereas those in the Fc
RIII·Fc interface are formed
primarily between the main-chain atoms or between the main-chain and
side-chain atoms. There are two salt bridges
Lys117-Asp362 and
Glu132-Arg334 observed between Fc
RI and Fc
but only one, Lys120-Asp265, is conserved in
the low affinity complex between Fc
RIII and Fc.
receptors (Fig.
5A). Three of them are also
conserved in the
-chain of Fc
RI. Gly159 in Fc
receptors is replaced with Trp in Fc
RI. Because Gly159
is in close contact with Leu235 from the lower hinge of
Fc-B, replacement of this residue with Trp may result in the observed
difference of the lower hinge conformation in IgE. Three other
interface residues, Lys120, Tyr132, and
Val158, are nearly invariant among all human Fc receptors.
The limited variation observed can be easily modeled into the existing
interface without creating steric hindrance. It is interesting that the interface salt bridge between Lys120 and Asp265
of the Fc appears to be absent in Fc
RI but conserved in all other
Fc
receptors and in Fc
RI. The other six interface residues, Ile88, Asp129, His134,
His135, Arg155, and Lys161 are less
well conserved among the receptors. Of these, variation at
His134 and His135 may result in conformational
changes in the lower hinge region of bound Fc. Overall, key features of
the receptor·Fc interface appear to be well preserved among all the
Fc receptors with possible hinge conformational adjustment for each
receptor·Fc pair. Of particular interest is the comparison between
the interface residues of Fc
RI and those of Fc
RIII. The binding
affinity of Fc
RIII is at least 100-fold weaker than that of Fc
RI.
Among the receptor·Fc interface residues, only four are different
between Fc
RIII and Fc
RI. These are Lys120,
Tyr132, Arg155, and Lys161 in
Fc
RIII and Asn120, Phe132,
Ser155, and His161 in Fc
RI. It is, however,
not clear if any of these residues contribute to the observed variation
in binding affinity.
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Fig. 5.
A, sequence alignment of the membrane
proximal domain of human Fc receptors. The secondary structure elements
(arrow for -strands and squiggle for
-helices) are indicated under the sequence. Residues identical to
the sequence of Fc
RIIIB are shown by periods, and gaps in
sequence are shown by minus signs. Residues contacting the A
and B chains of the IgG1 Fc are highlighted in red and
blue boxes, respectively. The predicted N-linked
glycosylation sites are indicated by asterisks.
B, sequence alignment of the lower hinge and CH2
regions of human IgG1, 2, 3, and 4, a mouse IgG2a and a human IgE C
3
region. The residues contacting Fc
RIII are highlighted in
red and blue boxes for A and B chains of the Fc,
respectively.
receptors display
IgG subtype specificities. In particular, human Fc
RIII binds tighter
to IgG1 and IgG3 than it does to IgG2 and IgG4. Most of the Fc residues
in contact with the receptor are conserved among the IgG sequences
(Fig. 5B, residues boxed in blue and
red), suggesting a conserved binding site for all human
IgGs. These binding residues, with the exception of a
Glu269 to Asp replacement, are also conserved in murine
IgG2a consistent with it being a ligand for human Fc
receptors. The
sequence differences among the IgG subclasses exist primarily at the
lower hinge region. First, hIgG2 has a Val and Ala at positions 234 and
235, respectively, instead of Leu and Leu as observed in IgG1 and IgG3,
and a one-residue deletion at position 237 of the corresponding IgG1.
Human IgG4 has a Phe at position 234 (Fig. 5B). In addition,
IgG2 and IgG4 sequences contain a three-residue deletion relative to
IgG1 at the N-terminal end of the lower hinge, possibly restricting the lower hinge conformation. The length of the lower hinge has been suggested as a factor in lower receptor binding affinity of IgG2 and
IgG4 (2). Among the four IgG subtypes, the length of the hinge region
is longest in IgG3 and shortest in IgG2 and IgG4 (three to four
residues shorter than that of IgG1). The differences in both the amino
acid composition and the length of lower hinge may contribute to the
observed lower receptor binding affinity of IgG2 and IgG4.
Receptor·Fc
Binding--
Both Fc
receptors and antibodies are glycosylated
in vivo. In contrast to the Fc fragment that displays only
one conserved carbohydrate attachment site located at
Asn297, the receptor glycosylation sites vary both in
number and in location among different Fc
receptors. For example,
the glycosylation sites on the C-terminal domain of Fc
receptors are
located at residues Asn162 and Asn169 on
Fc
RIII, Asn138 and Asn145 on Fc
RII and
asparagines 138, 145, and 149 on Fc
RI (Fig. 5A). The
influence of glycosylation on the receptor·Fc binding kinetics and on
the receptor function has been studied extensively using both the
deglycosylated receptor and Fc (4, 39). These studies demonstrated that
the carbohydrate attached to Asn297 of Fc have a
significant impact on the receptor binding, whereas glycosylations on
the receptors appeared less critical and perhaps have more of a
modulating effect on the affinity. For example, the two neutrophil
antigen A alleles of Fc
RIIIB, NA1 and NA2, differing primarily in
their carbohydrate contents, display a 2-fold difference in their
affinity for IgG3.
RIII-IgG Recognition--
On a cell surface, the
Fc receptor recognizes intact immunoglobulins. The presence of the Fab
portion of antibody is likely to impose restrictions to the
receptor·Fc recognition. To date, the only structure of an intact
antibody available is that of a mouse IgG2a (31). Because Fc
RIII
also recognizes mouse IgG2a, a model of this receptor·antibody
complex was generated by superimposing the Fc part of the current
structure onto the Fc of the IgG2a (Fig.
6A). This receptor·antibody
recognition model reveals that the receptor fits tightly and is
nearly engulfed by the bound antibody.
View larger version (41K):
[in a new window]
Fig. 6.
Antibody-Fc RIII
binding and ligand induced receptor aggregation model.
A, an intact antibody-Fc
RIII binding model. The structure
of the antibody is shown in magenta and that of Fc
RIII is
in green. The position of the second possible orientation of
Fc
RIII, which is in direct steric conflict with the hinge region and
Fab, is indicated by a blue-shaded area. The
arrow points to the location of the lower hinge
(L.H.). The Protein Data Bank entry for the antibody
coordinates is 1IGT. B, a simple avidity model of
antigen-antibody binding induced Fc
RIII aggregation. C,
an ordered receptor aggregation model.
receptor recognition, the mechanism of receptor activation, namely the
antigen-driven receptor clustering, remains unknown. Two receptor
clustering models can be proposed based on the current structural
results, a simple avidity model and an ordered receptor aggregation
model (Fig. 6, B and C). The simple avidity
receptor activation model assumes that the binding of oligomeric
antigens by antibodies increases the avidity as well as the proximity
of the receptors, which is sufficient to its activation. The ordered receptor aggregation model assumes that the binding of oligomeric antigens leads to the formation of an ordered receptor-ligand aggregation, which further stabilizes the activation complexes. Recent
imaging studies on T cell and NK cell receptor activation processes
suggest that the formation of the so-called immune synapse is an
ordered event (41, 42). These results favor the structured aggregation model rather than the simple avidity model, although the
molecular organization of Fc
receptors during their activation remain to be determined. Recently, an ordered receptor·ligand aggregate was observed in the crystal lattice of a natural killer cell
receptor in complex with its class I major histocompatibility complex
ligand (43). Such a receptor·ligand aggregate is not observed in the
two forms of the current Fc
RIII·Fc crystals. However, a parallel
receptor aggregate was observed in the crystal lattice of Fc
RIII in
the absence of Fc (22). A superposition of the current complex
structure onto this lattice receptor aggregate suggests that the
clustering model be compatible with the structure of a receptor·Fc
complex (Fig. 6C).
Receptor with Other Ligands of Fc--
The Fc
region of the IgG molecule possesses multiple recognition sites for
different components of immune system, including Fc
receptors,
neonatal Fc receptor (FcRn), rheumatoid factors (RF) and components of
the complement system. In addition, it is also used as a ligand by
staphylococcal proteins A and G. The structures of Fc complexed to
FcRn, RF, protein A, and protein G are now known (30, 33, 34, 44). The
binding of Fc by Fc
receptors is characteristically different from
all other known Fc ligands. First, the location of the Fc receptor
binding site differs from those of neonatal Fc receptor, RF, and
protein A. Although Fc
receptors bind to the lower hinge region of
Fc between the CH1 and CH2 domains, FcRn, RF,
protein A, and protein G bind to the joint region between the
CH2 and CH3 domains of Fc. Second, Fc
receptors recognize Fc in an asymmetric fashion resulting in one
receptor bound to both chains of Fc whereas all other ligands bind Fc
in a symmetric fashion with each chain of Fc harboring an intact
binding site (Fig. 7). The distinct
binding site for Fc
receptors suggests that it is possible to bind
Fc
receptors simultaneously with other ligands that recognize the
CH2-CH3 joint region on the same Fc molecule.
This raises the possibility of activation of multiple immune
components by the same antigen-bound immune complex.
View larger version (51K):
[in a new window]
Fig. 7.
Recognition of Fc by multiple ligands.
Structural comparison among the complexes of (A)
Fc RIIIB-Fc; (B) FcRn-Fc (PDB entry 1FRT); (C)
rheumatoid factors RF-Fc (PDB entry 1ADQ); and (D) bacterial
protein A-Fc (PDB entry 1FC2). Protein G binds similar to Fc as does
protein A. Due to its symmetric interaction with ligand, only one chain
of Fc is shown in the FcRn·Fc, RF·Fc and protein A·Fc complexes.
The corresponding Fc regions are colored in cyan and shown
in similar orientations. The ligands to Fc are colored in
green and the
2-microglobulin of FcRn is
shown in red. Only the variable domain of RF is shown. The
carbohydrates are shown in ball-and-stick models.
receptor signaling, namely the
need to have 1:1 recognition stoichiometry and to be capable of
discriminating the IgG subtypes. Both CH2 and
CH3 domains of Fc are very conserved among the subclasses
of IgGs. Even the CH2-CH3 joint region, which
is involved in binding of other Fc ligands, has near identical
sequences among the IgG subclasses (Fig. 5B). The lower
hinge region of IgGs, in comparison, is more variable allowing
subtype-specific recognition of the receptor. The conformation of the
hinge region, however, is quite flexible compared with the
CH2 and CH3 domains of Fc (35). This hinge flexibility, which enables the Fab arms to adapt to the shape and form
of antigens, may in fact hinder the binding of Fc receptors. Interestingly, there are two conserved cysteine residues forming two
disulfide bonds at the N-terminal end of the lower hinge. The presence
of these disulfides may stabilize the lower hinge conformation while
allowing sufficient flexibility at the upper hinge region. Finally, the
binding to the lower hinge region of both chains of Fc allows the
receptor to monitor the integrity of the antibody.
Rs, in
particular Fc
RI and Fc
RIII, also mediate the inflammatory
responses generated by cytotoxic autoantibodies and immune complex
triggered inflammatory disorders (45, 46). They provide a critical link
to autoimmune diseases, such as rheumatoid arthritis, hemolytic anemia,
and thrombocytopenia. The structure of Fc
RIIIB in complex with
IgG1-Fc reveals the molecular interface of this receptor·Fc
recognition and thus provides new possibilities for developing
therapeutic reagents to block the activation of Fc receptors by
autoantibodies. For example, the lower hinge sequence of Fc may be used
to generate neutralizing antibodies that could block the binding of
autoantibodies to Fc
Rs. The peptides encompassing residues of the BC
and FG loops of the C-terminal domain of Fc
receptors could also be
used to develop neutralizing antibodies against the receptors. Finally,
reagents that affect the glycosylation pathway may be used to affect
the carbohydrate composition of Fc and thus the conformation of the
receptor binding epitope.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. C. Hammer and M. Garfield for mass spectrometry measurements and N-terminal amino acid sequencing; Dr. J. Boyington and Dr. Z. Dauter for their assistance to the x-ray data collection at the National Synchrotron Light Source X9B beam line; and Dr. S. Ginell for his assistance at the Argonne National Laboratory Structural Biology Center 19-ID beamline at the Advanced Photon Source, whose use was supported by the U. S. Department of Energy, Office of Biological and Environmental Research, under Contract No. W-31-109-ENG-38.
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FOOTNOTES |
---|
* This work was supported by the intramural research funding of NIAID, National Institutes of Health and by INSERM, Institut Curie, France.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.
To whom correspondence should be addressed: Structural Biology
Section, Laboratory of Immunogenetics, NIAID, National Institutes of
Health, 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-496-3230; Fax: 301-402-0284; E-mail: psun@nih.gov.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100350200
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ABBREVIATIONS |
---|
The abbreviations used are:
FcRI and Fc
RI, Fc receptors for IgG and IgE of the immunoglobulin superfamily;
r.m.s., root mean square;
FcRn, neonatal Fc receptor;
RF, rheumatoid
factors.
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