# **Crystal Structure of a Human Fc Receptor in Complex with an Fc Fragment of IgG1**

Sergei Radaev^1^, Shawn Motyka^2^, Wolf-Herman Fridman^3^, Catherine
Sautes-Fridman^3^, and Peter D. Sun^1\*^

^1^Structural Biology Section, Laboratory of Immunogenetics, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, 12441 Parklawn Drive, Rockville, MD 20852,USA

^2^Biochemistry, Cellular and Molecular Biology Program, Johns Hopkins
University, School of Medicine, 725 N. Wolfe Street, Baltimore, MD
21205,USA

^3^Laboratory of Cellular and Clinical Immunology, INSERM Unit 255,
Curie Institute, Paris, France

\* Corresponding author: <psun@nih.gov>; Phone: 301-496-3230; Fax:
301-402-0284.

Running title: the crystal structure of FcRIII-Fc complex

***Summary***

Fc gamma receptors mediate antibody dependent inflammatory response and
cytotoxicity as well as certain autoimmune dysfunction. Here we report
the crystal structure of a human Fc receptor (FcRIIIB) in complex with
an Fc fragment of human IgG1. The C-terminal domain of FcRIII binds
asymmetrically to the lower hinge region of both chains of Fc. No major
conformational changes are observed in both the receptor and Fc upon
complex formation. Removal of the carbohydrate attached to the conserved
glycosylation residue Asn 297 on Fc drastically reduced the receptor
binding affinity suggesting a role for the carbohydrate in providing the
receptor epitope conformational stability. A model of antibody-FcRIII
interaction supports the role of C~H~1-C~H~2 hinge length in determining
the IgG isotype specificity. The structure suggests novel ways of
designing therapeutic reagents against autoimmune diseases, such as
rheumatoid arthritis.

## *Introduction*

Fc receptors, which are expressed on the majority of haematopoietic
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 (ADCC). The three major types of Fc receptors are Fc, Fc
and neonatal Fc receptors. Except for the neonatal Fc receptor and FcRII
(CD23), which are related structurally to class I major
histocompatibility antigens and C-type lectins, respectively, all other
known Fc receptors are members of immunoglobulin superfamily(Daeron,
1997; Tamm and Schmidt, 1997). Among them, FcRI and FcRI 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 FcRII and FcRIII, are low affinity receptors with dissociation
constants ranging from 10^-5^ to 10^-7^ M(Hulett and Hogarth, 1994;
Galon et al., 1997; Powell et al., 1999). In addition to variations in
affinity, each receptor displays distinct IgG subtype specificities.
Unlike the high-affinity receptors that can be activated by monomeric
antibodies, the low-affinity receptors preferentially respond to the
activation by immune complexes.

Human FcRIII exists as two isoforms, FcRIIIA and FcRIIIB that share 96%
sequence identity in their extracellular immunoglobulin binding regions.
FcRIIIA is expressed on macrophages, mast cells and natural killer cells
as a transmembrane receptor. In contrast, FcRIIIB, present exclusively
on neutrophils, is anchored by a glycosyl-phosphatidylinositol (GPI)
linker to the plasma membrane. While FcRIIIA associates with the
immuno-tyrosine activation motif (ITAM) containing FcRI -chain or the T
cell receptor -chain for its signaling, FcRIIIB lacks a signaling
component. Nevertheless, it plays an active role in triggering Ca^2+^
mobilization and in neutrophil degranulation (Kimberly et al., 1990;
Unkeless et al., 1995). In addition, FcRIIIB, in conjunction with
FcRIIA, activates phagocytosis, degranulation, and the oxidative burst
that leads to the clearance of opsonized pathogens by neutrophils.
Recently, a soluble form of FcRIIIB was reported to activate the CR3
complement receptor dependent inflammatory process (Galon et al., 1996).

The Fc binding region on FcRII and FcRIII has been identified through
the work of chimeric receptors with FcRI 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(Hulett et al., 1994; Hulett et al., 1995; Tamm et
al., 1996). Similar regions on the -chain of FcRI were also identified
to be critical for IgE binding affinity (Cook et al., 1997). The
receptor binding site on Fc has been located through the construction of
chimeric IgG and mutational analysis at the lower hinge region, residues
located in the hinge region between C~H~1 and C~H~2 domains and
immediate adjcent to the N-terminus of the C~H~2 domain of IgG (Chappel
et al., 1991; Canfield and Morrison, 1991; Lund et al., 1991). 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 FcRI binding (Henry et
al., 1997). Apart from the lower hinge region, a few residues on the
C~H~2 domain of an IgG2b were also suggested to interact with the
receptor(Kato et al., 2000). However, with the exception of the neonatal
Fc receptor, the molecular recognition between the Fc receptors and Fc
remains to be elucidated(Burmeister et al., 1994a).

# The recent crystal structures of a FcRI, FcRIIA and FcRIIB revealed a conserved immunoglobulin-like (Ig-like) structure, particularly the small hinge angle between the two Ig-like domains, that is unique to the Fc receptors (Garman et al., 1998; Sondermann et al., 1999; Maxwell et al., 1999). We report here the crystal structure of a human FcRIII in complex with Fc portion of a human IgG1 determined from two crystal forms.

###### *Results and Discussion*

## Overall structure of the complex

Crystals of a human FcRIII receptor in complex with a human Fc fragment
of IgG1 were grown in two forms under different conditions. The
orthorhombic crystals belonged to the space group of P2~1~2~1~2~1~ and
diffracted to 3.3Å resolution where as the hexagonal crystals had
P6~5~22 space group symmetry and diffracted to 3.5Å 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
R~cryst~ =22.9% and R~free~ =27.3% for the orthorhombic form and
R~cryst~ =24.9% and R~free~ =32.6% for the hexagonal form,
respectively(Table 1). The electron density is continuous throughout the
complex in the final 2*F~o~*-*F~c~* map except for three surface loops
of FcRIII (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
forms contain one FcRIII and one Fc molecule in each asymmetric unit
suggesting a 1:1 stoichiometry for the binding between the receptor and
Fc (Figure 1). This is consistent with earlier binding studies using
non-equilibrium and equilibrium gel filtration experiments(Zhang et al.,
2000). The structure of FcRIII-Fc complex observed in both crystal forms
is essentially identical.

**The structure of FcRIII**

The structure of FcRIII 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(Figure 2a)(Zhang et al., 2000).
The hinge angle between the N-terminal (D1) and the C-terminal (D2)
domains is 60°, slightly larger than the 53° value observed in the
ligand free receptor. However, no significant change in the receptor
conformation is observed upon complex formation (Figure 2a).

## 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, C~H~2
and C~H~3. The overall shape of the Fc fragment resembles that of a
horse shoe with the two C~H~3 domains packing tightly against each other
at the bottom of the horse shoe and the C~H~2 domains held apart by
carbohydrate moieties attached to the glycosylation site Asn 297 from
both chains forming the opening of the horse shoe. Well defined electron
density throughout the Fc allowed for unambiguous tracing of residues
A234 to A444 in chain A and B232 to B443 in chain B of Fc, including the
lower hinge regions, A234-A238 and B234-B238. The structure of the Fc
fragment in complex with FcRIII does not differ significantly from that
observed in the structures of unbound Fc fragment and a mouse intact
IgG2a antibody(Deisenhofer, 1981; Harris et al., 1992)(Figure 2b).
However, the two-fold symmetry relating the two chains of Fc in other
unligated Fc structures, is no longer retained in the structure of the
complex. The horse shoe shaped Fc is slightly more open at the
N-terminal end of the C~H~2 domains in the FcRIII-Fc structure compared
to other known structures of Fc. The hinge angle between C~H~2 and C~H~3
domains of chain A (Fc-A) is 95° and 100 in the orthorhombic and the
hexagonal crystals, respectively, approximately 10 larger than the
corresponding angle of chain B (Fc-B) and the 84°- 89° angle observed in
all structures of ligand-free Fc (Figure 2b).

## The interface between FcRIII and Fc

The receptor binds to Fc at the center of the horse shoe 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) (Figure 1). Such a 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 suggesting the receptor-Fc recognition stoichiometry be 1:1.
The receptor-Fc complex buries approximately 1453 Å ^2^ solvent
accessible area (Figure 3a). The interface between FcRIII and Fc
molecules shows poor shape complementarity with mean shape correlation
statistic of 0.53(Lawrence and Colman, 1993), less than those between
T-cell antigen receptor and Class I MHC 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 (Figure 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 horse shoe opening making contacts to 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 FcRIII
protrudes into the opening between the two chains of Fc (Figure 1a). All
three receptor loops (BC, C'E and FG) were implicated in Fc binding
through earlier studies of chimeric FcRII /FcRI receptors and through
site directed mutagenesis(Hulett et al., 1994; Hulett et al., 1995; Cook
et al., 1997). On the Fc side of the complex, interactions with the
receptor are dominated by residues Leu 234-Pro 238 of the lower hinge
(Table 2), consistent with the results form earlier mutational work
(Tamm and Schmidt, 1997). In particular, Hinge-A and B together
contribute approximately 60% of the overall receptor-Fc interface area
(Figure 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 (Deisenhofer, 1981; Sauer-Eriksson et al., 1995; Corper et
al., 1997; Harris et al., 1997). In contrast, residues of both Hinge-A
and B are clearly visible in the electron density maps of both crystal
forms suggesting that the binding of FcRIII stabilizes the lower hinge
conformation of Fc.

A combination of salt bridges, hydrogen bonds and hydrophobic
interactions contributes to the receptor-Fc recognition. Specifically,
the interface between FcRIII and Fc-A is dominated by hydrogen bonding
interactions whereas the hydrophobic interactions occur primarily at the
interface between the FcRIII 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 (Figure 3, Table 2). Seven of which are distributed across
the receptor and Fc-A interface and two of which are at the receptor and
Fc-B interface. Alanine mutations, such as H134A mutant of FcRII 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 (Hulett et al., 1995). A hydrophobic core is formed between Trp
90, Trp 113 of the receptor and Pro B329 of the C~H~2 domain of Fc-B
(Figure 3c). This hydrophobic core extends further to include Val 158,
the aliphatic side chain of Lys 161 of the receptor and Leu B235 of
Hinge-B. Mutations of both Trp 113 and Lys 161 in FcRIII lead to the
loss in receptor function (Hibbs et al., 1994; Tamm et al., 1996). The
side chain of Leu B235 packs tightly against Gly 159 of the receptor
leaving little space to accommodate any residues larger than Gly at this
position. A G159A mutation on chimeric FcRII resulted in the disruption
of Fc binding presumably due to the steric hindrance between Leu B235
and the Alanine mutant at position 159(Hulett et al., 1994). Of
particular interest is Trp 113 of the receptor, that when mutated to Phe
resulted in the loss of Fc binding. This residue not only is 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 FcRIII. W113F mutation would lead to the change in
inter-domain conformation and the disruption in binding as the result of
loss in this wedge which restricts the inter-domain hinge angle
movement.

## Conserved receptor-Fc binding interface

Of the 13 receptor interface residues, four (Trp 90, Trp 113, Lys 131
and Gly 159) are invariant among all human Fc receptors (Figure 4a).
Three of them are also conserved in the -chain of FcRI. Gly 159 in Fc
receptors is replaced with Trp in FcRI. Since Gly 159 is in close
contact with Leu B235 from the lower hinge of the Fc-B, replacement of
this residue with Trp is likely to result in readjustment in the lower
hinge conformation of IgE. Three other interface residues, Lys 120, Tyr
132 and Val 158, 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 Lys 120 and Asp A265 of the Fc appears to
be absent in FcRI but conserved in all other Fc receptors and in FcRI.
The other six interface residues, Ile 88, Asp 129, His 134, His 135, Arg
155 and Lys 161 are less well conserved among the receptors. Of these,
variation at His 134 and His 135 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 the particular interest is the comparison between
the interface residues of FcRI and those of FcRIII. The binding affinity
of FcRIII is at least 100 fold weaker than that of FcRI. Among the
receptor-Fc interface residues, four are different between FcRIII and
FcRI. They are Lys 120, Tyr 132, Arg 155 and Lys 161 in FcRIII and Asn
120, Phe 132, Ser 155 and His 161 in FcRI. It is, however, not clear if
any of these residues contribute to the observed variation in binding
affinity.

# **Fc Receptor IgG subtype specificities**

All Fc receptors display IgG subtype specificities. In particular, human
FcRIII binds human IgG1 and IgG3 much tighter than IgG2 and IgG4. The
ten receptor recognition residues, six on one chain of Fc and four on
the opposing chain, are conserved among the four IgG subtypes with the
only exception of an alanine replacement of leucine at position 235 in
IgG2 (Figure 4b). With the exception of a Glu 269 to Asp replacement,
these interface residues are also conserved in mouse IgG2a, consistent
with it being recognized by human FcRIII. The presence of invariant
interface residues in IgG4 compared to that of IgG1 suggests factors
other than the composition of the interface residues contribute to the
observed difference in binding affinity. The length of lower hinge, and
thus its flexibility has been suggested as a factor in lower receptor
binding affinity of IgG2 and IgG4(Tamm and Schmidt, 1997). In the
current FcRIII-IgG2b model, the placement of FcRIII did create minor
steric clashes with the Fab arm above it requiring an adjustment in the
hinge to reposition the Fab in order to accommodate the receptor
binding. These results are consistent with flexibility of the hinge
being a contributing factor in receptor affinity. Among the four IgG
subtypes, the length of the hinge region is longest in IgG3 and shortest
in IgG2 and IgG4 (three to four residue shorter than that of IgG1). This
difference in the length of the hinge is likely a more important factor
in determining receptor-IgG subtype affinity than the residues at the
receptor-Fc interface and the lack of hinge flexibility in IgG2 and IgG4
due to their shorter length may contribute to the lower receptor binding
these IgG subtypes.

# **The contribution of carbohydrate to the Fc 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 Asn 297, the receptor glycosylation sites
varies both in the number and in the location among different Fc
receptors. For example, the glycosylation sites on the C-terminal domain
of Fc receptors are located at residues Asn 162 and 169 on FcRIII, Asn
138 and 145 on FcRII and Asn 138,145, and 149 on FcRI (Figure 4a). 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 (Galon et al., 1997; Jefferis et al.,
1995). These studies demonstrated that the carbohydrate attached to Asn
297 of Fc had a significant impact on the receptor binding whereas
glycosylations on the receptors appeared less critical and perhaps more
important for modulating the affinity. For example, the two neutrophil
antigen A alleles of FcRIIIB, NA1 and NA2 differing primarily in their
carbohydrate contents display a two-fold difference in their affinity
for IgG3.

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 Asn 297 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 Asn 297 of both chains of
Fc toward each other into the inter-chain region, referred to as the
carbohydrate core region. This unique arrangement between the
oligosaccharide moieties and the polypeptide chains of Fc suggests that
carbohydrate may provide conformational stability for the receptor
binding epitopes (Jefferis et al., 1998). Specifically, the spacing and
the orientation between the two C~H~2 domains may be influenced by the
presence of sugar attachments (Figure 5a). Since 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 C~H~2 domains.

The binding of the Fc fragment and the intact IgG1 to FcRIII was
measured using BIACore 2000 solution binding experiments. Approximately
15 µM concentration of FcRIII was immobilized on a CM5 sensor chip via
primary amine attachment (Biacore,Inc). The binding experiments were
performed using a serial dilution of Fc or IgG1 between 10 and 0.02 µM
concentration range. The binding of both Fc and IgG1 to the receptor
displays fast on and off kinetic rate constant (k~on~ \> 10^3^
M^-1^s^-1^, k~off~ \>0.3 s^-1^). The dissociation constant for Fc and
IgG1 binding to FcRIII were 4.4 and 4.0 µM, respectively, consistent
with previously published results (Galon et al., 1997; Zhang et al.,
2000). Deglycosylation of Fc by peptide-N-glycosidase F (PNGase F, New
England Biolabs) resulted in a molecular weight shift from a
heterogeneous profile with average of 53400 to 50345 Daltons measured by
electrospray mass spectrometry(Figure 5a). The molecular weight of
deglycosylated Fc agrees well the predicted polypeptide mass of 50407,
suggesting a complete removal of carbohydrate moieties attached to the
Fc. The deglycosylated Fc remained dimeric and retarded slightly
compared to the native Fc on size exclusion chromatography (Figure 5b).
Similar molecular weight shift was observed in the deglycosylation of
IgG1. The binding for the deglycosylated Fc and IgG1to FcRIII in
solution was, however, 10-20 fold weaker (Table 3), suggesting a
critical role for the carbohydrate attached to Asn 297 of Fc in the
receptor-ligand recognition. Since the carbohydrate moieties on the Fc
are not in direct contact with the receptor, it suggests that their
effect on the receptor binding is likely conformational. That is, the
deglycosylation of an antibody may disrupt the respective orientation of
the two lower hinge loops and thus result in a loss of recognition by
the receptor.

The glycosylation on the receptor appears less critical to the Fc
binding. The recombinant FcRIII used in this work was produced from
*E.coli* and lacks carbohydrate modification but was nevertheless
capable of binding to the Fc. Moreover, most of the receptor
glycosylation sites are located away from the receptor-Fc interface
making it unlikely for them to contribute to the Fc binding affinity.
The only exception is Asn 162, a potential glycosylation site observed
in FcRIII but not in other Fc receptors. Asn 162 is located at the end
of the FG loop in the D2 domain of FcRIII. The protrusion of the FG loop
into the Fc opening results in Asn 162 directly above the carbohydrate
core of Fc with its side chain oriented toward the carbohydrate (Figure
5a). This may create a direct contact between the oligosaccharide
moieties from Asn 162 of the receptor and the oligosaccharide moieties
from Asn 297 of the Fc allowing the modulation of the receptor-Fc
recognition through the content of carbohydrate.

# **Model for FcRIII-IgG recognition** 

On a cell surface, 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 (Harris et al., 1992). Since
FcRIII 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. Due to the existence of a two-fold
symmetry between the two chains of Fc, there are two orientations in
which the current FcRIII-Fc complex structure can be superimposed onto
that of IgG2a. This results in two non-equivalent binding orientations
of the receptor on IgG2a owing to the asymmetrical orientations of the
two Fab regions and of the lower hinge region with respect to that of
Fc. One binding orientation positions the receptor on one side of the
lower hinge and directly underneath one of the Fab arm (Figure 6). The
second orientation places the receptor at the location occupied by the
hinge between the Fab and Fc creating severe steric clashes with both
the hinge and the Fab. It thus appears that Fc receptors can only
recognize antibodies in one orientation. This receptor-antibody
recognition model also reveals that the receptor fits tightly and is
nearly engulfed by the bound antibody.

## Recognition of Fc by multiple ligands

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 protein A and protein G. The structures of Fc complexed
to FcRn, RF, protein A and protein G are now known (Burmeister et al.,
1994b; Corper et al., 1997; Deisenhofer, 1981; Sauer-Eriksson et al.,
1995). The binding of Fc by Fc receptors is characteristically different
from that by neonatal Fc receptor (FcRn), RF, protein A and by protein
G. First, all other known Fc ligands bind Fc in a symmetric fashion with
each chain of Fc harboring an intact binding site (Figure 7). In
contrast, Fc receptors recognize Fc in an asymmetric fashion resulting
in one receptor bound to both chains of Fc. Second, the location of the
Fc receptor binding site also differs from those of neonatal Fc
receptor, RF, and protein A. While Fc receptors bind to the lower hinge
region of Fc between the C~H~1 and C~H~2 domains, FcRn, RF, protein A
and protein G bind to the joint region between the C~H~2 and C~H~3
domains of Fc. The distinct binding site for Fc receptors suggests that
it is possible to bind Fc receptors simultaneously with other ligands
that recognize the C~H~2 - C~H~3 joint region on the same Fc molecule.
This raises the possibility of activation of multiple immune components
by the same antigen-bound immune complex. The integrity of the Fc
receptor binding interface depends on the specific orientation of both
chains of Fc and thus becomes potentially sensitive to the
conformational changes. This creates a unique situation in which the
glycosylation of Fc may influence the receptor binding by altering the
conformation of Fc.

## Receptor-IgG recognition and autoimmune diseases

In addition to their normal cellular functions in host immunity, FcRs,
in particular FcRI and FcRIII, also mediate the inflammatory responses
generated by cytotoxic autoantibodies and immune complex triggered
inflammatory disorders (Ravetch and Clynes, 1998; Boros et al., 1994).
They provide a critical link to autoimmune diseases, such as rheumatoid
arthritis, hemolytic anemia and thrombocytopenia. The structure of
FcRIIIB 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 FcRs. 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
affect the glycosylation pathway may be used to affect the carbohydrate
composition of Fc and thus the conformation of the receptor binding
epitope.

As the structure of the receptor-Fc complex helps us to understand the
molecular recognition and the function of this receptor-ligand pair, it
also leaves many questions unanswered. For example, the molecular
interactions responsible for the binding affinity differences of FcRI
and FcRIII remain to be determined. The role of carbohydrate in the
receptor-Fc recognition needs to be further clarified. Finally, the
activation of Fc receptors requires cross-linking by antigens on cell
surface regardless the receptor to Fc affinity. In the absence of
antigen, binding of immunoglobulins to Fc receptor does not lead to the
receptor aggregation as is in the situation of high affinity FcRI and
FcRI receptors which exist mostly in IgG or IgE bound form on cell
surface. In deed, the complex structure described here precludes the
binding of second receptor molecule on the Fc therefore eliminates the
possibility of receptor aggregation by Fc binding alone and prevents the
potential deleterious effect of antibodies. Thus, the structure of a 1:1
receptor-Fc complex ensures the need of antigen in forming the receptor
aggregation, suggesting that the requirement of antigen bound
immune-complexes in the activation of low affinity receptors is not only
an avidity issue but also a mechanism for receptor aggregation and thus
the activation of the immune system.

# ***Experimental Procedures***

## Protein expression, purification and crystallization

The extracellular part of human FcRIIIb receptor, residues 1-172, was
expressed as *Escherichia coli* inclusion bodies and then reconstituted
in vitro as described(Zhang et al., 2000). Fc fragments of human IgG1
antibody were prepared by the papain digestion as previously reported
(Kato et al., 1991a).(Kato et al., 1991b)

The complex of Fc and FcRIII was prepared by mixing both components in
1:1 molar ratio and was concentrated to 8-15 mg ml^-1^ for
crystallization. Single crystals of two forms - orthorombic and
hexagonal were obtained by vapor diffusion in hanging drops at room
temperature under slightly different crystallization conditions.
Rod-shaped crystals of the orthorombic form were grown from 10% PEG4000
and 50 mM Hepes at pH 6.5. They appeared after 2-3 days and grew to
average size of 0.05×0.05×0.2 mm in approximately two weeks. Crystals of
the second form, hexagonal bipyramids, were crystallized from 5%PEG6000
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 one month.

## Structure determination

After brief 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
CCD detector at the X9B beam line of the National Synchrotron Light
Source (NSLS) at the Brookhaven National Laboratory and processed with
HKL2000(Otwinowski and Minor, 1997). The hexagonal crystals belong to
space group *P*6~5~22 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 *P*2~1~2~1~2~1~ with cell
dimensions of *a*=76.4, *b*=102.8 and *c*=123.3 Å diffracted to 3.3 Å.
Both crystal forms contain one molecule of FcRIII and one molecule of Fc
in each asymmetric units.

The structure of the FcRIII-Fc complex in both crystal forms was
determined by molecular replacement. Polyalanine models of Fc (PDB
accession number 1FC1) and FcRIII 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 Fc molecule was determined in both crystal forms using AmoRe
(Navaza, 1994). 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 (R~f~) of 51%
(CC=51% and R~f~=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 R~f~=52% (CC=46%
and R~f~=50% after rigid-body refinement in AmoRe). The position of
FcRIII was determined in both crystal forms using the program EPMR
(Kissinger et al., 1999) with a polyalanine model of FcRIII and the
position of the Fc molecule fixed. Clear solutions were obtained for
both crystal forms with CC=56% and R~f~=48% for orthorombic crystal and
CC=55% and R~f~=48% for hexagonal crystal, respectively. After
rigid-body refinement of individual domains of the FcRIII-Fc complex
modeled as polyalanine using CNS (Brunger et al., 1998) 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 v0.9. Model adjustments and rebuilding were done using the
program O (Kleywegt and Jones, 1996). Carbohydrate molecules were added
manually using 2*F~o~*-*F~c~* electron density maps contoured at 1.0and
refined. The final model includes residues 5-172 of FcRIII, residues
235-444 for one chain of Fc and residues 233-443 for the second chain of
Fc.

#### Surface plasmon resonance measurements

Deglycosylated Fc fragment and IgG1 were prepared by PNGase F (New
England Biolabs) digestion in water for 1.5 h at 37°C (**unit enzyme/mg
protein ???).** The extend of deglycosylation were analyzed by SDS-PAGE,
gel filtration chromatography and electrospray ionization mass
spectrometry. The gel filtration experiments were carried out using a
Superdex 200 PC 3.2/30 gel filtration column (Amersham Pharmacia
Biotech) and an Äkta HPLC purifier (Amersham Pharmacia Biotech) with 50
mM NaCl, 50 mM Tris at pH 8.0 as running buffer at a 0.1 ml/min flow
rate. Electrospray ionization mass spectrometry (ESI-MS) measurements
were acquired and recorded with a Perkin-Elmer Sciex API-300 triple
quadruple system (Thornhill, Ontario, Canada) using 8 g the native or
deglycosylated Fc fragment, 6 g the native or deglycosylated IgG1. The
samples of IgG1 and its deglycosylated form were reduced with 100 mM DTT
prior mass spectrometry measurements.

Surface plasmon resonance (SPR) measurements were performed using a
BIAcore 2000 instrument (BIAcore AB). Soluble FcRIII receptor (0.3
mg/ml) was immobilized on a CM5 sensor chip at pH 6.0 with
*N*-hydrosuccinimide / 1-ethyl-3(-3-dimethylaminopropyl)-carbodiimide
hydrochloride (NHS/EDC). Solutions containing either the Fc fragment or
the IgG1 in 20 mM NaCl, 3mM EDTA, 0.005% surfactant P20, 10 mM Hepes at
pH 7.4 were used as analyte. Binding of the native and deglycosylated Fc
fragments of IgG1 to the immobilized FcRIII was measured using serial
dilutions of the analyte from 10 to 0.078 M at a flow rate of 5 l/min.
The binding of the IgG1 and its deglycosylated form to the receptor was
measured with the analyte concentration varying from 10 to 0.02 M. The
dissociation constant (*K*~d~) was obtained from either a steady-state
curve fitting analysis using BIAevaluation 3.0 (BIAvore AB) or linear
regression using ORIGIN 3.0 (MicroCal Software, Inc.).

**Acknowledgement**

The authors wish to thank Drs. 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 NSLS X9B beam line; Ms. A. Hanson for secretarial
assistance; This work was supported by National Institute of Allergy and
Infectious Diseases, National Institutes of Health, USA and by INSERM,
Institut Curie, France.

Added note: During the preparation of this manuscript, two related
structures of Fc and their receptor complexes were published (Garman et
al., 2000; Sondermann et al., 2000).

Table 1. Data collection and refinement statistics

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Data Collection Orthorhombic Form Hexagonal Form

Space group *P*2~1~2~1~2~1~ *P*6~5~22

# Unit cell parameters (Å) *a*=76.4, *b*=102.8, *c*=123.3 *a*=*b*=114.9, *c*=301.4

Resolution limit (Å) 3.3 3.5

Unique reflections 13216(1286)\* 15541(1511)

Redundancy 3.0(2.8) 4.7(4.8)

Completeness (%) 86.4(86.1) 98.7(99.4)

*R*~sym~ (%)^†^ 12.5(46.5) 8.7(42.3)

*I*/(*I*) 9.7(2.5) 20.2(4.0)

Refinement

Resolution (Å) 10-3.3 10-3.5

No. reflections 11948 14032

No. nonhydrogen atoms 4733 4712

No. atoms in carbohydrate

moieties 195 149

*R*~cryst~(%) 22.9 24.9

*R*~free~(%)^‡^ 27.3 32.6

Mean B-factor (Å ^2^) 41 67

Wilson B-factor (Å ^2^) 54 82

r.m.s.d. bond lengths (Å) 0.008 0.008

r.m.s.d. bond angles (°) 1.49 1.51

\*Values for highest resolution shells, 3.30-3.44 Å for orthorhombic and
3.50-3.60 Å for hexagonal forms, are shown in parentheses.

^†^*R*~sym~=100*I~h~*-\<*I~h~*\> /*~h~*, where \<*I~h~*\> is the mean
intensity of multiple measurements of symmetry equivalent reflections.

^‡^*R*~free~ is calculated using 3% of the reflections in the test set.

**Table 2. Interface contacts between FcRIII and Fc**

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

# 

# Hydrogen bonds and salt bridges

FcRIII Fc Distance (Å)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Thr116 O~1~ LeuB235 O 3.3

Lys120 N Ser A239 O 3.4

N Asp A265 O~1~ 3.2

N Gly A237 O 3.4

Tyr132 OH Asp A265 O~2~ 3.8

His134 N~1~ Gly A237 O 3.6

His134 N~2~ Gly A236 O 3.5

His134 N~2~ Gly A237 O 2.4

His135 N~2~ Leu A235 O 3.2

Val158 O Gly B236 N 3.5

Lys161 N Gly B236 O 3.0

Hydrophobic contacts\*

Ile88 AlaB330

Trp90 ProB329, GlyB236

Trp113 ProB329

His134 GlyA236, GlyA237, AspA265

Val158 LeuB235

Gly159 LeuB235

Lys161 GlyB236

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\*Carbon-carbon contacts 4.0 Å.

**Table 3. Dissociation constant for the binding of FcRIII and Fc**

Analyte\* K~D~ (M)\*\*

Flow Cell 2 Flow Cell 3

Fc Native 4.6 6.7

#  Deglycosylated \> 200 \> 150

IgG1 Native 4.8 3.1

Deglycosylated 69.2 43.3

\*FcRIII are immobilized to a CM5 sensor chip in flow cell 2 and 3 via
primary amine attachment at approximately concentrations of 15 and 45 M,
respectively. Each binding experiment was carried out with a serial 2x
dilution of 8 to 10 concentration points of the analyte starting at 10
M.

\*\*The dissociation constant for the binding of the native Fc and IgG1
(K~D~^n^ ) to the CM5 chip was estimated from the steady state curve
fitting. The dissociation of deglycosylated Fc and IgG1 (K~D~^d^ ) was
estimated from the equation :

Req^d^ / Req^n^ = (K~D~^n^ + C) / (K~D~^d^ + C)

where Req and C are equilibrium response unit and the concentration of
analyte.

**Figure Legends**

Figure 1. Ribbon drawing showing front view (a) and side view (b) of the
FcRIII-Fc. The side view of the complex is rotated 90° from the front
view. FcRIII is shown in cyan, A and B chain of Fc are painted yellow
and green, respectively. Positions of D1 and D2 domains of FcRIII as
well as C~H~2 and C~H~3 domains of Fc are marked. Carbohydrate moieties
are shown in grey.

Figure 2. (a) Superposition of the structure of FcRIII in the
receptor-Fc complex (in cyan) with that of ligand free receptor (in
red). (b) Superposition of the structure of Fc in FcRIII-Fc complex ( in
green) with that of unbound Fc (in magenta).

Figure 3. FcRIII-Fc interface (a) Surface representation of FcRIII.
Interface region is colored. Regions involved in the interactions with
Hinge-A and Hinge-B are painted yellow and green, respectively. All
other contact areas are colored in blue. Lower hinge regions of Fc are
in ball and stick representation. (b) Interactions between FcRIII (cyan)
and chain A of Fc (yellow). The glycosylation residue, Asn A297, is also
shown. (c) Interactions between FcRIII (cyan) and chain B of Fc (green).
The side chain of Val 158 is omitted for clarity. There is a hydrogen
bond between carbonyl group of Val 158 and backbone nitrogen of Gly B236
(table 2) 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 FcRIII which
play an important role in the interactions are labeled. Some secondary
structure elements of Fc lying behind and not contributing in the
binding shown semi-transparent. Carbohydrate moieties has been omitted
for clarity.

Figure 4 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 FcRIIIB are shown by (.) and gaps in
sequence are shown by (-). 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 (\*). b)
Sequence alignment of the lower hinge and C~H~2 regions of human
IgG1,2,3, 4, a mouse IgG2a and a human IgE C3 region. The residues
contacting FcRIII are highlighted in red and blue boxes for A and B
chains of the Fc, respectively.

Figure 5 (a). Carbohydrate attachment on the Fc. The A and B chains of
Fc are shown in orange and yellow, respectively. The glycosylation site,
Asn 297, is shown in ball and stick model on both chains of Fc. FcRIII
is shown is cyan. Residues in the vicinity of the glycosylation sites
are labeled. The attached carbohydrate moieties are represented by green
ball and stick model with oxygen atoms in red.

Figure 6. A model of FcRIII binding to an intact antibody. The structure
of the antibody is shown in orange and that of FcRIII is in green. The
position of the second possible orientation of FcRIII, 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.

Figure 7. Recognition of Fc by multiple ligands. Structural comparison
among the complexes of (a) FcRIIIB-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 coloured in green and shown in similar
orientations. The ligands to Fc are coloured in cyan 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.

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