# **Crystal Structure of the Ligand-binding Domain of a Human FcRIII**

Yihong Zhang^1^, Christian C. Boesen^2^,Sergei Radaev^2^, Andrew G.
Brooks^3^, Wolf-Herman Fridman^4^, Catherine Sautes-Fridman^4^, and
Peter D. Sun^2\*^

^1^Department of Human Biological Chemistry and Genetics, University of
Texas Medical Branch at Galveston, Galveston, TX 77555

^2^Structural Biology Section, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, 12441 Parklawn
Drive, Rockville, MD 20852

^3^Dept of Microbiology and Immunology, University of Melbourne, Royal
Parade, Parkville, Victoria, 3052, Australia

^4^Laboratory of Cellular and Clinical Immunology, INSERM Unit 255,
Curie Institute, Paris, France

\* Corresponding author: psun@nih.gov

Running title: crystal structure of FcRIII (CD16)

# **Summary**

Fc receptors play a major role in immune defenses against pathogens and
in inflammatory processes. The crystal structure of a human
immunoglobulin receptor, FcRIIIb, has been determined to 2.3 Å
resolution. The overall fold consists of two immunoglobulin-like domains
with an acute interdomain hinge angle of 47 and 51 degrees,
respectively, for the two molecules in the asymmetric unit. Trp 113,
wedged between the N-terminal D1 and the C-terminal D2 domains, appears
to further restrict the hinge angle. The putative Fc binding region of
the receptor carries a net positive charge complementary to the negative
charged receptor binding regions on Fc. Two separate parallel dimers are
observed in the crystal lattice offering intriguing models for receptor
aggregation.

## Introduction

Because of their ability to bind the Fc portion of antibodies, Fc
receptors provide a link between humoral and cellular immune responses.
There are Fc receptors for each Ig isotype. Except for FcRII (CD23)
which is C-type lectin like, all other known Fc receptors are members of
immunoglobulin superfamily (Daeron, 1997). Among them, FcRI, and FcRI
are high affinity receptors with dissociation constants ranging from
10^-8^ to 10^-10^ M, whereas the other receptors, 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).

FcRIII, also known as CD16, is the type III Fc receptor. It exists as
two isoforms in human, FcRIIIa and FcRIIIb sharing 96% sequence identity
in their extracellular immunoglobulin binding regions. FcRIIIa is
expressed on macrophages, mast cells, natural killer cells as a
transmembrane receptor. Present exclusively on neutrophils, FcRIIIb is
the only Fc receptor anchored by a glycosyl-phosphatidylinositol (GPI)
linker to the plasma membrane. Despite the lack of a signaling
component, FcRIIIb plays an active role in triggering Ca^2+^
mobilization and neutrophil degranulation (Kimberly et al., 1990;
Unkeless et al., 1995). In addition, in conjunction with FcRIIa, FcRIIIb
activates phagocytosis, degranulation, and oxidative burst which lead 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
immunoglobulin-binding affinities of FcRIIIb were reported to be
1x10^-6^ and 2x10^-6^ M for human IgG1 and IgG3, respectively (Galon et
al., 1997).

# 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., 1999a; Maxwell et al., 1999). Several models of receptor activation / oligomerization have been proposed based on the crystal lattice packings and molecular modeling. However, discrepancies exist among these models and none appear to form an oligomeric state. We report here a 1.8 Å crystal structure of a human FcRIIIb and its implication to the receptor oligomerization based on our observed crystal lattice packing. 

## Results and Discussion

## Overall structure of FcRIII

The sequence of human FcRIII is 49% identical to FcRIIa and 39%
identical to FcRI (Figure 1). Two forms of the FcRIII receptor crystal
were obtained. The structure was initially determined on the lower
resolution crystal form to 2.3 Å resolution by multiwavelength anomalous
dispersion (MAD) method using a HgCl~2~ derivative (Table 1). The
refined R-values are 0.24 and 0.29 for R~cryst~ and R~free~,
respectively for this crystal form. The two receptor molecules, A and B,
in each crystallographic asymmetric unit were refined independently
without non-crystallographic symmetry (NCS) constraints. A second form
of the receptor crystal, which relates to the first crystal form by
converting the non-crystallographic two-fold into a crystallographic
two-fold symmetry, was obtained under slightly different crystallization
conditions. The refined receptor model from the first crystal form was
further refined to 1.8 Å resolution in the second crystal form with the
final R values of 0.18 and 0.21 for R~cryst~ and R~free~, respectively.
The refined model contains amino acid 3-175 of the mature sequence of
FcRIII ligand binding domain. The electron density is well defined
through out the molecule in refined (2Fo-Fc) map (Figure 2). The overall
structure of FcRIII consists of two immunoglobulin-like domains (Figure
3), similar to those of FcRIIa, FcRIIb and FcRI(Garman et al., 1998;
Sondermann et al., 1999a; Maxwell et al., 1999). Different from the
C2-like hematopoietic growth factor receptor fold, the first -strand of
both domains contains a switch from A to A', pairing with strand B and
G, respectively. This strand switch is centered at Pro 14 and Pro 96 of
the D1 and D2 domains and is also observed in the structure of
killer-cell immunoglobulin-like receptors (KIR)(Fan et al., 1997; Snyder
et al., 1999; Maenaka et al., 1999). Pro 14 is in the *cis-*
conformation whereas Pro 96 is in the *trans-*conformation and can be
replaced by a Ser at this location as is in the structure of FcRI and in
the sequence of FcRI. The structure of the N-terminal D1 domain of
FcRIII can be readily superimposed to the C-terminal D2 domain,
resulting in a root-mean-square (rms) difference of 1.2 Å between 73
pair of C atoms.

The refined structures of the two receptor molecules in the first
crystal form are nearly identical to each other and to that of the
second crystal form with the differences among the individual domains
less than 0.7 Å in root-mean-square (r.m.s.) deviation.

Among members of known Ig superfamily receptors, the structure of Fc
receptors is most similar to that of killer-cell immunoglobulin-like
(KIR) receptors despite the existence of only 18% sequence identity.
They not only share the same fold, but the individual domains can be
readily superimposed, resulting in rms differences of 1.9 and 1.5 Å
among their individual D1 (72 superimposed C atoms) and D2 (71
superimposed C atoms) domains, respectively.

## The inter-domain hinge angle

A feature common to all known Ig superfamily Fc receptors is their acute
hinge angle between the N- and C- terminal Ig domains. This angle was
reported between 52º and 70º for FcRIIa and FcRIIb, respectively, a
discrepancy most likely caused by the different algorithms used in
estimating the angles. The inter-domain hinge angle is calculated to be
51º, 47º and 49º for the receptors A, B of the first crystal form and
the receptor of the second crystal form, respectively. Compared to other
Ig-like structures, such as the hematopoietic receptors, KIR receptors
and adhesion receptors, Fc receptors not only have smaller hinge angles,
but the position of their N-terminal (D1) domain with respect to the
C-terminal (D2) domain is also opposite to other receptors (Figure 4a).
Detailed examination of the hinge region reveals the molecular basis for
the unique inter-domain hinge angle common to all Fc receptors. First,
as noted in the FcRIIa structure, inter-domain linker for all Fc
receptors consists of two residues, shorter than the five residues
linker observed in KIR and growth hormone receptor. The KIR like D1, D2
arrangement requires a longer inter-domain linker while the linker for
Fc receptors appears too short to adopt the KIR like inter-domain
arrangement. Second, the BC loop from the D2 domain, residues 113-118,
protrude away from the core of D2 domain toward the D1 domain instead of
assuming a typical up-and-down direction (Figure 4b) as is the BC loop
of the D1 domain. As a result, this BC loop acts as a cap situated atop
of the D1 domain restricting its position. A similar conformation is
also found in the BC loop from the structure of FcRI. Third, a conserved
tryptophan residue, Trp 113, (also referred to as Trp 110 in the
structures of FcRIIb and FcRI) (Figure 1), from the BC loop of the D2
domain inserts itself into one end of D1 domain as a wedge to further
restrict the hinge movement (Figure 4b). Any increase in the hinge angle
would create steric hindrance between Trp 113 and the backbone of the AB
loop in D1 domain. A Trp 113 to Ala mutation in FcRI, that would likely
result in a conformational change, has been shown to affect the IgE
binding dramatically (Hulett et al., 1999). Third, there are also
favorable interdomain interactions to stabilize the tight D1/D2 packing
(Table 2). One such interaction is a conserved salt bridge between Asp
23 of the D1 domain and His 111 of the D2 domain. There is also a small
core of hydrophobic residues, consisting of Trp 16, Leu 20, Leu 92, Gln
94 and Trp 113, in the hinge region. Hydrogen bonds and van der Waals
interactions among hydrophilic residues are also found in the hinge
region providing additional stability to the hinge angle. The D1, D2
interface buries 1290 Å^2^ solvent exposed area. The 7º hinge angle
difference between the molecule A and B is most likely the result of a
bound phosphate ion that is found in the hinge region of molecule B but
not in A (Figure 4c). The existence of a bound phosphate ion was also
evident from mass spectrometry of FcRIII (data not shown). It forms
hydrogen bonds with the main chain of Lys 114 and Thr 116, the side
chain of His 111. Although the biological relevance of the
phosphate-bound and phosphate-free forms of the receptor remains
unclear, the difference in hinge angle observed between the two
molecules in the asymmetric unit suggests that the hinge angle can vary.

## The putative ligand binding region

Extensive mutational analysis on Fc receptors in recent years has
identified regions of the receptors that are important for the binding
of immunoglobulins. In particular, the construction of FcRII and FcRI
chimeric receptors and the use of site directed mutagenesis have
identified residues on the BC, C'E and FG loops of the D2 domain
important for the function of Fc receptors (Hulett et al., 1994; Hulett
et al., 1995; Henry et al., 1997; Cook et al., 1997). However, the
details of molecular recognition between Fc and Fc receptors remain to
be elucidated. Structurally, these loops encompass a rather extensive
area and together with the hinge loop, residues 88-90, and the AB loop
of D1 domain, residue 21-23, they form a putative ligand binding region
(Figure 5a). There are a total of 16 residues in this region. With six
potential positively charged amino acids and only one negatively charged
amino acid, the putative ligand binding region of FcRIII appears
overwhelmingly positively charged. Interestingly, the sequence alignment
of the immunoglobulin Fc region shows that the presumed Fc receptor
binding site, the lower hinge region and the loops nearby, carries a net
negative charge (Figure 5b)(Kato et al., 2000). This results in an
apparent charge complementarity between the receptor and Fc.

Among Fc receptors, FcRI and FcRI are high affinity receptors whereas
FcRII and FcRIII are low affinity receptors. It has been proposed that a
Trp ladder observed in the structure of FcRI be critical to its ligand
affinity (Garman et al., 1998). In addition, the superposition of the
receptor D2 domains between FcRIII and FcRI shows that significant
structural differences in the BC, C'E and FG loops exist between the two
receptors while the -strands are closely superimposed. In particular,
the C'E loop differs as much as 5 Å between the two receptors, with the
loop adopting an up conformation in FcRIII and down conformation in
FcRI. It is possible that this conformational difference contributes to
part of the difference in ligand binding affinity.

#### Stoichiometry of Fc and FcRIII recognition

## Recent solution binding studies suggest the binding of IgG be 1:1 for both FcRIII and FcRII (Ghirlando et al., 1995; Kato et al., 2000), although both 1:1 and 2:1 receptor-ligand binding modes have been proposed (Metzger, 1992; Maxwell et al., 1999; Sondermann et al., 1999b). We have examined the binding of FcRIII with an Fc derived from a human IgG~1~ using both analytical ultracentrifugation (AUC) and size exclusion chromatography techniques. The affinity between the receptor and Fc measured by AUC experiment is 0.4 M. The receptor-ligand binding stoichiometry, however, could not be confirmed from the AUC experiments. To determine the receptor-ligand binding stoichiometry, we have used both the non-equilibrium and the equilibrium size exclusion chromatography techniques (Hummel and Dreyer, 1962; Sondermann et al., 1999b; Martin and Bjorkman, 1999). Under the non-equilibrium conditions, a series of gel filtration experiments were carried out using 10 M of Fc mixed with 10, 20 and 30 M of FcRIII to form the 1:1, 2:1 and 3:1 receptor:Fc molar ratio of the complex (Figure 6a). The apparent molecular weights of the receptor, Fc and their complex are 25, 51 and 63 killodaltons, respectively. The appearance of excess receptor in 3:1 and 2:1 but not 1:1 molar ratio is consistent with a 1:1 receptor:ligand binding stoichiometry. The composition of FcRIII and Fc in the complex peak was also estimated by N-terminal peptide sequencing. The result is also consistent with a 1:1 molar ratio between FcRIII and Fc. 

#### The equilibrium gel filtration experiments were performed with the column equilibrated using a buffer containing 10 M FcRIII (Figure 6b). A trough corresponding to that of the receptor was observed when the sample contained only Fc (at 10 M concentration), indicating a depletion of the receptor in running buffer. The trough disappeared at the receptor-ligand ratio of 1:1 and developed into a peak at the receptor: ligand ratio of 2:1 and 3:1. Similar gel filtration profiles were also observed, when the column was equilibrated with 1 M of Fc instead of the receptor. The results from both the non-equlibrate and equilibrate gel filtration experiments suggest a 1:1 receptor:ligand stoichometry.

## Receptor dimerization and aggregation in crystal 

Ligand induced receptor aggregation is presumed to be a common mechanism
for initiating receptor mediated signaling. Structurally, one of the
best known examples is the human growth hormone induced receptor
dimerization in which one molecule of growth hormone or erythropoietin
binds to two molecules of their receptors(de Vos et al., 1992; Livnah et
al., 1999). The situation between Fc and Fc receptors is less clear due
to the lack of the structural information regarding to the
receptor-ligand complex. In the first crystal form of FcRIII, there are
two receptor molecules, A and B, in each crystallographic asymmetric
unit. As a result, we observed two different dimeric packings among
symmetry related molecules. (Figure 7a,b,c). The first dimer is formed
between molecule A or B and their translational related molecules in
adjacent unit cells, the AA or BB dimer (Figure 7a,b). They are
identical parallel dimers, in which monomers of the same orientation
associate through a D1/D2 interaction. The interface of this AA dimer
consists of portions of the B and E -strands from D1 domain of one
molecule and portions of the F and G -strands from the D2 domain of
another molecule. The interface buries 860 Å^2^ solvent exposed surface.
The putative ligand binding regions in this dimer are in identical
orientation. The predicted glycosylation sites are located outside the
dimer interface. Since the monomers are related by translational
symmetry, the dimer so generated extends much beyond one unit cell. In
fact they form an ordered oligomeric aggregation throughout the crystal
in which each receptor links with a proceeding receptor via its D1
domain and a successive receptor via its D2 domain using the same form
of D1/D2 interface (Figure 7a). The second dimer, the AB dimer, observed
in the crystal of FcRIII is formed between receptors A and B in each
asymmetric unit. This is also a parallel dimer but with a different
interface than in the AA and BB dimers (Figure 7c). The interface of AB
dimer is formed between the C' strand of the receptor A D2 domain and
the C, C' strands of the receptor B D1 domain. It buries 500 Å^2^
solvent exposed surface. Like the AA or BB dimer, the predicted
glycosylation sites of the receptor are located outside the interface
area. An interesting characteristic common to both AA, BB and AB dimers
is their parallel rather than two-fold relationships between the
monomers. Since this parallel symmetry is incompatible with the two fold
symmetry of an immunoglobulin constant Fc region, it predicts that the
parallel dimers are consistent only with a 1:1 receptor:ligand
stoichiometry. The surface charges of FcRIII are rather non-uniformly
distributed with the N-terminal D1 domain mostly negatively charged and
the C-terminal D2 domain mostly positively charged (Figure 5b). This
charge distribution may also facilitate the D1/D2 dimerization.

The existence of FcR oligomers on the cell surface remains to be proven.
However, since FcRII and FcRIII have a low affinity for monomeric IgG,
it is tempting to speculate that oligomerization of receptor allows the
immune complexes to interact simultaneously with several Fc receptors.
This would increase the avidity of IgG-FcR interaction and facilitate
the triggering of cellular responses. Figure 7d depicts a hypothetical
receptor aggregation model with a 1:1 stoichiometry based on the
parallel dimer observed in our current crystal. Different from any
two-fold symmetry related dimer, the parallel dimer aggregation model
has the capability to polymerize and form a more extensive oligomeric
receptor-ligand state.

## Experimental Procedures

### Protein expression and purification

The extracellular ligand binding domain of a human FcRIIIb, residues
1-172 of the mature sequence, was subcloned into a Novagen pET-28b
vector using the NcoI and NotI restriction sites and a E. coli BL21(DE3)
strain. Two additional amino acids ( Met-Ala) were added to the 5' end
of the gene and a histidine tag (Ala-Ala-Ala-Leu-Glu-His~6~) was added
to the 3' end to facilitate the expression and purification (Snyder et
al., 1999). The protein was first expressed in an inclusion body form
and then reconstituted in vitro. In brief, cells containing the FcRIIIb
expressing plasmid were grown in a 10 liter Newbrunswick Bioflo 3000
bio-reactor vessel and induced with 0.5 mM IPTG at an approximate O.D.
595 of 10.0 for 5 hours. Once harvested, the inclusion bodies were
isolated by repeated wash with 2 M urea solutions and then re-dissolved
in 8 M urea prior to refolding. The refolding reaction was initiated by
a quick dilution of the urea dissolved inclusion bodies of FcRIIIb into
a refolding buffer consisting of 0.5M arginine, 5mM CaCl~2~, 5mM
cysteamine, 5mM cystamine, 10 ug/ml AEBSF and 50 mM Tris/pH 8.0, and
then dialyzed thoroughly against water. The re-natured FcRIIIb was
concentrated on a Ni-NTA affinity column and further purified on a
Superdex 200 size exclusion column. The integrity of the refolded
receptor was analyzed both by N-terminal amino acid sequencing and by
electrospray ionization mass spectrometry (ESI-MS).

#### *Analytical Ultracentrifugation experiments* 

#### Sedimentation equilibrium profiles were obtained in a Beckman XL-A, using absorbance optics at wavelengths of 230, 250 and 280 nm. 150ul of samples in 5mM phosphate buffer, at different concentrations of Fc and CD16 were used at rotor speeds of 14,000 rpm, and 18,000 rpm. Sedimentation equilibrium profiles of both CD16 and the Fc dimer were well described by that of thermodynamically ideal monomeric species, with buoyant molar masses consistent with those calculated from the amino acid sequence. Analysis of the mixtures was performed by a global fit of several equilibrium distributions at multiple rotor speeds, protein concentrations, and molar ratios.

# *Non-equilibrium and Equilibrium Gel Filtration Experiments*

For non-equilibrium gel filtration experiments, FcRIII was incubated
with Fc (Fc concentration was kept at 10 M) at 1:1, 2:1, and 3:1 molar
ratios in a running buffer of 50 mM Tris, pH 8.0, 50 mM NaCl for at
least 30 min. A sample of 25 l was injected onto a Superdex 200 PC
3.2/30 gel filtration column (Amersham Pharmacia Biotech) equilibrated
with the same buffer at a flow rate of 0.1 ml/min. The absorbance of the
eluent was monitored at 280 nm and fractions in 0.1 ml volumn were
analyzed by SDS-PAGE. N-terminal peptide sequencing by Edman degradation
method was used to quantify the amount of FcRIII and Fc in the isolated
receptor-Fc complex.

For equilibrium gel filtration experiments, the Superdex 200 PC 3.2/30
gel filtration column (Amersham Pharmacia Biotech) was first
equilibrated with a buffer containing 50 mM Tris, pH 8.0, 50 mM NaCl and
10M FcRIII. Samples (25 l) of molar compositions 10M FcRIII:10M Fc, 15M
FcRIII:10M Fc, 20M FcRIII:10M Fc, and 30M FcRIII:10M Fc were incubated
at room temperature in the same buffer for at least 30 min before
injection onto the column at a flow rate of 0.1 ml/min.

### Crystallization, data collection and structure determination

Two forms of FcRIII crystals were obtained by hanging drop methods. The
first form was grown from a buffer of 10% PEG 4000, 50mM Sodium HEPES at
pH 7.0 and a protein concentration of approximately 7 mg/ml. Crystals
grew to a size of 0.1 x0.2 x0.5mm^3^ in about one month and diffracted
to 2.3 Å resolution. A HgCl~2~ derivative was identified using a mass
spectrometry assisted heavy atom screening method (Sun and Hammer,
2000). The mercury derivative crystal was prepared by soaking crystals
in a well solution containing 0.5% saturated HgCl~2~ for 12 hours. Both
the native (Native_1) and the mercury derivative data were collected at
the Brookhaven NSLS X9B beamline under cryofreezing temperature (-180 C)
and processed using HKL2000 (Table 1) (Otwinowski and Minor, 1997). The
crystals belong to a space group P2~1~2~1~2 with the unit cell
dimensions of a= 78.9, b=147.8, c=36.4 Å and two molecules in each
asymmetric unit. Multi-wavelength anomalous dispersion (MAD) data were
collected at the L~III~ edge of Hg (Table 1). The heavy atom binding
sites were determined by both inspecting of the difference Patterson
maps and by using the SHELX program and refined by using the program
MLPHARE (Sheldrick, 1990; Otwinowski, 1994). The initial chain tracing
and all subsequent model building were done using the program O version
6.2.1 (Kleywegt and Jones, 1996). The structure was refined with CNS
version 0.9 using a maximum likelihood target function (Brunger et al.,
1998) and without NCS constraints. The refinement started with rigid
body refinement for each domains and followed by alternating positional
and grouped B-factor refinement. Maps were calculated and model was
revised using the program O at the end of each round of positional and
B-factor refinement. After all protein residues were built and refined,
water molecules were added to peaks higher than 2-3 in the residual
difference map and with likely hydrogen bonding partners. The second
form of FcRIII crystals was obtained under slightly different condition
with 10% PEG 8000 and 50 mM sodium Hepes at pH 6.5 as the precipitation
reagent. The crystals belong to the same space group of P2~1~2~1~2 as
the first form but with different cell dimensions of a=67.4, b=85.7 and
c=36.3 Å and only one receptor molecule in the crystallographic
asymmetric unit. A native data (Native_2) was collected on an in-house
Raxis IV imaging plate detector to 1.8 Å resolution. The interdomain
hinge angle is defined as the angle between the principal ellipsoid axes
of the D1 and D2 domains of the receptor and is calculated using the
program HINGE (Snyder et al., 1999).

#### Acknowledgement

The authors wish to thank Drs. C. Hammer and M. Garfield for mass
spectrometry measurements and N-terminal amino acid sequencing; Dr. P.
Schuck for performing the analytical ultracentrifugation measurements;
Dr. Z. Dauter for the assistance during the X-ray data collection at the
X9B beam line in NSLS; Dr. B. Seed for the gift of FcRIIIb cDNA. This
work was supported by National Institute of Allergy and Infectious
Diseases, National Institutes of Health, US and by INSERM, Institut
Curie, France.

Figure Legends

Figure 1. Sequence alignment of human Fc receptors. Amino acids
identical to the sequence of FcRIIa are not shown. The secondary
structure elements of FcRIII are shown above the sequence with D1 domain
in black and D2 domain in gray. Residues involved in the inter-domain
hinge packing are boxed in red. Residues in the putative Fc binding
region are shown in magenta.

Figure 2. A representive section of the refined ( 2Fo-Fc ) map of FcRIII
at 1.8 Å resolution.

Figure 3. Structure of FcRIII. The N-terminal D1 and the C-terminal D2
domains are colored in blue and green, respectively. The putative ligand
binding loops are colored in red. The strands for D1 domain are A
(5-12), A'(17-20), B(22-33), C(39-45), C'(47-52), E(55-61), F(66-73),
G(77-85) and the strands for D2 domain are A(91-95), A'(98-101),
B(104-113), C(120-125), C'(130-134), E(136-143), F(149-157) and
G(163-172). Residues 146-149 form a short -helix. This and the
subsequent ribbon figures are prepared using the program MOLSCRIPT and
Raster3D (Kraulis, 1991; Merritt and Bacon, 1997).

Figure 4 (a) Relative orientation of the D1 and D2 domains in KIR and
FcRIII. The D2 domains of KIR and FcRIII are superimposed and their
orientations are kept identical in the figure. (b) The inter-domain
hinge region. The D1, D2 domain of FcRIII and their respective
interdomain packing residues are shown in dark blue and green. The BC
loop of D2 domain is colored in orange. The dashed line indicates the
salt bridge between Asp 23 and His 111. The superposition of D1 domain
on the D2 domain is shown in light blue and its BC loop in purple. (c)
Superposition of the molecule A and B of FcRIII in the asymmetric unit.
A bound phosphate ion is found in B but not in A.

Figure 5 (a) The putative Fc binding region. The D1 and D2 domains of
FcRIII are shown in dark blue and green, respectively. Residues in the
putative ligand binding region of FcRIII are shown in ball-and-stick
model. The D2 domain of FcRI superimposed onto the D2 domain of FcRIII
is shown in light blue. (b) The Grasp representation of the
electrostatic potential distribution at the putative interface of FcRIII
and the C~H~2 domain of IgG. Positive potentials are colored in blue and
negative potentials are in red. The putative interface regions are
highlighted. The coordinates for Fc are taken from the structure of
IgG2a, Protein Data Bank entry 1IGT.

Figure 6. Analysis of the FcRIII and Fc complex by size exclusion
chromatography under both non-equilibrium and equilibrium conditions.
The apparent molecular weights of FcRIII, Fc and their complex are 25,
51 and 63 kilodaltons, respectively. The predicted molecular masses for
FcRIII and Fc are 21.0 and 50.4 kilodaltons, respectively. (a)
Non-equilibrium gel filtration. The injection sample contains 10 M
FcRIII or 10 M Fc for the FcRIII and Fc only runs. The complexes were
prepared with 10 M Fc and various amount of the receptor to give rise
the 1:1, 2:2 and 3:1 receptor:Fc molar ratio. (b) Equilibrium gel
filtrations were carried out using a buffer containing 50 mM NaCl, 50 mM
Tris pH 8.0 and 10M FcRIII. Samples contain 10M Fc with various amount
of FcRIII as indicated in each curve. The elution volumes of FcRIII and
the FcRIII:Fc complex are marked.

Figure 7. Fc receptor dimerization and aggregation model. (a) Crystal
lattice packing of molecule A (in red) and B (in green). (b) AA or BB
parallel dimers. The dimers are formed through translational symmetry.
The view is 90° from that of panel (a). D1 and D2 domains are colored in
light blue and green. The interface residues are shown as
ball-and-sticks in dark blue and green for D1 and D2 domains,
respectively. The predicted glycosylation sites are shown in magenta.
The putative Fc binding loops are colored red. (c) AB parallel dimer.
The dimer is formed between the two molecules in each asymmetric unit.
(d) A hypothetical model of receptor aggregation based on the observed
parallel AB dimer (in green) in crystal lattice. The antibody
coordinates, colored in magenta and blue, are from Protein Data Bank
entry 1IGT.

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