From the
Ministry of Education Protein Science
Laboratory & Laboratory of Structural Biology, Tsinghua University,
Beijing, 100084, China, the ||Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical
College, 5 Dong Dan San Tiao, Beijing 100005, China, the
Institute of Biophysics, Chinese Academy of
Science, Beijing 100101, China, and the **Division of
Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810
Sapporo, Japan
Received for publication, May 27, 2003 , and in revised form, June 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Five types of IgA receptors have been recognized so far. They are
FcRI (CD89), the polymeric Ig receptor, Fc
/µR, the
transferrin receptor, and the asialoglycoprotein receptor
(1). Among them, Fc
RI is
the only one that specifically binds IgA. On ligation of IgA complexed with
antigens, Fc
RI is able to mediate various cellular responses including
phagocytosis, antibody-dependent cell cytotoxicity, oxidative bursts, and
release of inflammatory mediators
(1).
FcRI belongs to the immunoglobulin superfamily and contains an
extracellular region of 206 amino acids, a transmembrane domain of 19 amino
acids and a cytoplasmic region of 41 amino acids
(4). The extracellular region
of Fc
RI consists of two Ig-like domains, EC1 and EC2, and six potential
sites for N-glycosylation. The receptor binds IgA1 and IgA2 with an
equal affinity (5). A number of
residues including Tyr35, Arg52, Tyr81,
Arg82, Ile83, Gly84, His85, and
Tyr86 on Fc
RI are potentially involved in IgA binding
(6,
7).
Although FcRI is an immunoglobulin Fc receptor
(FcR),1 it differs in
many ways with FcRs for other immunoglobulin classes. IgG receptor
Fc
RIII and IgE receptor Fc
RI bind antibodies in the near hinge
regions and form 1:1 complexes
(8,
9), whereas Fc
RI binds
the CH2-CH3 interface of Fc
(10,
11) and preferably forms 2:1
complex with a single Fc
homodimer
(12). It has been reported
that Fc
Rs and Fc
RI use their membrane proximal-domain and linker
region binds immunoglobulin (8,
9,
13,
14), whereas Fc
RI uses
its membrane-distal domain EC1 to bind IgA
(15).
The genes of most FcRs are located in chromosome 1 at 1q21
[PDB]
23
(16), whereas FcRI is
in chromosome 19, at 19q13.4
(17,
18), a region called the
leukocyte receptor complex because it is clustered with several leukocyte
receptor families including killer cell inhibitory receptors (KIRs) and
leukocyte Ig-like receptors (LIR/LILR/ILTs)
(17,
18). The amino acid sequence
of Fc
RI shares only 20% homology with other FcRs, but it has around 35%
homology with its neighboring LIRs and KIRs
(1).
In this paper, we report our analysis of the crystal structure of the
ectodomain of FcRI expressed in Escherichia coli and its
comparison with FcRs, LIR, and KIR.
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EXPERIMENTAL PROCEDURES |
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Crystallization, Data Collection, and Structure
DeterminationThe FcRI crystals were obtained by the
hanging-drop method. The crystals were grown from a buffer of 11.4%
polyethylene glycol-8000 in 100 mM sodium Hepes buffer, pH 7.6,
containing 8% (v/v) ethanol glycol, 3% (v/v) Me2SO, and 50
mM MgCl2 as an additive, and protein concentration of
12 mg/ml. The Se-Met derivative crystal was grown from the same
conditions. The Se-Met derivative data were collected at the Spring8 beamline
BL41XU under 100 K at wavelengths 0.9798 Å, 0.9800, and 0.9000 Å
and processed using HKL2000
(19). The crystals belong to
the space group C2221 with the unit cell dimensions of a =
59.0, b = 69.5, c = 106.4 Å and one molecule in each
asymmetric unit. The SOLVE program
(20) was used to locate Se
sites and to calculate initial phases. Following density modification by
RESOLVE (21), the resultant
electron density map was of sufficient quality that the entire model except
for one flexible loop and several residues at the termini could be built. The
initial chain tracing and all subsequent model building were done using the
program O (22), version 8.0.
Refinement was performed using CNS1.0
(23) and merged synchrotron
data with Fobs > 0. The Bijvoet pairs of the data used
in refinement are unmerged. The model was initially refined as a rigid body
with data 8.04.0 Å resolution. The resolution was extended
gradually, and subsequent refinement used protocols including anisotropic
temperature factor refinement, energy minimization, and slow cool simulated
annealing. Several rounds of manual refitting using omit maps permitted the
missing loop regions to be traced and side chains built. 68 water molecules
were built into the electron density when a Fo
Fc map, contoured at 3.5
,
coincided with well defined electron density of a
2Fo Fc map
contoured at 1
. The N-terminal 2 additional residues (MA), C-terminal
15 residues (DSIHQDYTTQNLILE), and residues 5659 (FWNE) were disordered
in the crystal. The final model contained 191 residues of Fc
RI and 68
solvent molecules. Rcryst and Rfree
were 0.210 and 0.239, respectively, for data in the resolution range
40.02.1 Å. The structure contains two cis prolines at
position 154 and 161. None of the main-chain torsion angles are located in
disallowed regions of the Ramachandran plot. Statistics for data collection,
phasing, and refinement are shown in Table
I.
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For analyses of interdomain angles, contacts, and buried surface areas, D1 was defined as residues 1100 and D2 was defined as residues 101195, following the structure-based definition of KIR2DL1 domain boundaries (24). Interdomain contact residues were defined as being within 3.6 Å of the partner domain and identified using CONTACT (35). Buried surface areas were calculated using SURFACE (35) with a 1.4-Å probe radius.
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RESULTS AND DISCUSSION |
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In the EC1 domain, four anti-parallel -strands (A, B, E, D) oppose a
sheet of five
-strands (C',C,F,G,A')
(Fig. 1b). There is a
-bulge (Ser91 to Thr93) in the G strand of EC1
splitting the strand into two short
-strands.
The EC2 domain is built up from eight -strands arranged such that
three stands (A, B, E) form one
-sheet and five strands (C', C, F,
G, A') form a second
-sheet. EC2 does not have a strand in the
corresponding position to strand D of EC1. Three residues (Tyr181,
Leu182, and Trp183) on EC2 F-G loop stick out. Two of
them (Tyr181 and Trp183) form hydrogen bonds with
Val98 on EC1 and the OH group of the side chain of
Tyr181 forms another hydrogen bond with the side chain of
Glu95. Furthermore, Trp183 forms hydrogen bonds with
Gly100 on the end of EC1 and Leu101 on the linker.
The interdomain angle of EC1 and EC2 is calculated to be 85°. The bent
shape of the FcRI produces a large interface between the D1D2 domains
that buries 1134 Å2 of the accessible surface area
(Fig. 1c). Most of the
residues involved in the EC1 and EC2 interdomain interaction are hydrophobic,
including Val17 on EC1 A' strand, Ala74 and
Gly75 on EC1 E-F loop, Val97, Val98,
Thr99, and Gly100 on EC1 G strand, Leu101 and
Tyr102 on the linker strand and Tyr173 on EC2 F strand,
Tyr181 and Trp183 on the EC2 F-G loop. The hydrophobic
core formed by interactions between these residues stabilizes the interdomain
angle. Five of them, namely Val17, Tyr97,
Tyr173, Tyr181, and Trp183, are most likely
to be important for the conformation. Hydrogen bonds are also found in the
hinge region providing additional stability to the hinge angle. These hydrogen
bonds (Glu95 O
-Tyr181 OH, Val98
N-Tyr181 O, Val98 O-Trp183 N,
Gly100 N-Trp183 O) mainly involve main-chain atoms and
are therefore independent of sequence variation.
As FcRI shows a relatively high degree of homology to the D1 and D2
domains of KIR and LIR, the C
atoms of these three receptors were
superimposed to analyze their structural similarities. As shown in
Fig. 2a, the overall
structures of the three receptors are similar especially for the EC2 and D2
domains. The major difference is found in the corresponding position of EC1 C,
C', and D strands of Fc
RI. In LIR-1 D1, strands C' and D
are replaced by two 310 helices. On the other hand, the C'
strand of Fc
RI is shorter than that of KIR2DL1 and KIR2DL2. Hence, the
C-C' loop in Fc
RI EC1 forms earlier
(Fig. 2b), allowing
the C-C' loop and F-G loop to adopt a clamp-like arrangement. A similar
feature can also be found in many other FcRs
(Fig. 2c) even though
they share low degree of identity with Fc
RI. The significance of this
is still unknown.
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Although LIR-1 does not contain of C' and D strands in the D1 domain,
it more closely resembles FcRI in the other part of the molecule
compared with KIR2DL1 and KIR2DL2. The root mean square deviation (r.m.s.d)
values for the C
atoms are 1.44 Å for Fc
RI EC1 and LIR-1
D1, 1.54 Å for Fc
RI EC1 and KIR2DL2 D1 and 1.77 Å for
Fc
RI EC1 and KIR2DL1 D1. It seems the lack of C' and D strands in
LIR-1 D1 have little effect on its overall structure, although the sequence of
C' and D regions are variable within KIR2DL1, KIR2DL2, and LIR-1
(Fig. 3).
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The interdomain angle of FcRI is closer to that of LIR-1 (84 to
90°) (25), but larger than
that of KIR2DL2 (60 to 80°)
(26). The hydrophobic core
interface observed in Fc
RI also exists in LIR-1 and KIR2DL2
(25,
26). Amino acid sequence
alignment shows the 12 hydrophobic residues, especially Tyr181 and
Trp183, which play an important role in stabilizing the interdomain
angle in Fc
RI are also conserved in LIR-1 and KIRs, having only one
residue (Leu101
Ala) different for LIR-1, three residues
(Val17
Leu, Val98
Ile, and
Tyr173
Phe) different for KIR2DL2 and four residues
(Val17
Leu, Val98
Ile, Thr99
Ile and Tyr173
Phe) different for KIR2DL1. These 12
residues are also conserved in a KIR from cow, with only one residue
(Tyr102
Ser) different from Fc
RI in this region
(Fig. 3). Moreover, a bovine
IgG2 FcR, Fc
2R, also possesses most of these hydrophobic residues and
only four residues (Val97
Leu, Thr99
Ala
and Tyr102
Arg and Trp183
Leu) are
different from Fc
RI. This Fc
2R has been previously found to
share more similarities to Fc
RI (41%) than to other types of human
Fc
Rs (less than 28%)
(27), and it is located on the
same chromosome as bovine KIR
(28,
29), indicating that it
belongs to the bovine leukocyte receptor complex. In contrast, such a
hydrophobic core does not exist in human FcRs for IgG and IgE
(11,
3033).
This suggests that the hydrophobic core is a common feature of receptors from
the leukocyte receptor complex and Fc
RI is evolutionally closer to LIR
and KIR than to other human FcRs.
As an immunoglobulin Fc receptor, FcRI differs from other FcRs not
only in structure but also in its ligand binding characteristics. IgG receptor
Fc
RIII and IgE receptor Fc
RI bind antibodies in the near hinge
regions and form 1:1 complexes
(8,
9). In contrast, Fc
RI
binds the CH2-CH3 interface of Fc
(10,
11) and preferably forms a 2:1
complex with a single Fc
homodimer
(12). It has been reported
that Fc
Rs and Fc
RI use their membrane-proximal domain and linker
region to bind immunoglobulin
(8,
9,
13,
14), whereas Fc
RI uses
its membrane-distal domain EC1 to bind IgA
(33). A number of residues
have been implicated in IgA binding, including Tyr35,
Arg52, Tyr81, Arg82, Ile83,
Gly84, His85, and Tyr86
(6). The crystal structure of
Fc
RI shows that Tyr35 is located in the B-C loop,
Arg52 is located in C' strand, Tyr81 and
Arg82 are located in F strand, and the remainder are in the F-G
loop. All these residues lie on the receptor surface except Tyr81,
which is buried inside the receptor and is unlikely to be involved directly in
IgA binding (Fig. 4).
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FcRI has six potential N-linked glycosylation sites
(Asn44, Asn58, Asn120, Asn156,
Asn165, and Asn177). Unglycosylated Fc
RI has a
molecular mass of 30 kDa. When expressed in vivo, its molecular mass
is increased to 50100 kDa due to different degrees of glycosylation
(1). Although the effect of
glycosylation still needs to be elucidated, carbohydrates seem to play an
important role in IgA binding since desialylated Fc
RI binds five times
more strongly to IgA (1).
Fig. 4 shows the position of
potential N-linked glycosylation site at Asn44, which is
close to the docking sites of IgA.
In conclusion, the crystal structure and sequence alignment show that
FcRI is a member of the leukocyte receptor complex and evolutionally
closer to LIR than KIR. All members of this complex found so far share a
common hydrophobic core structure. The crystal structure also locates the
residues that are involved in Fc
RI binding to IgA.
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FOOTNOTES |
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* This work was supported by grants from the National Natural Science
Foundation of China (No. C03020
[GenBank]
50102), Natural Science Foundation of Beijing
(No. 7012026), Project "863" (No. 2001AA233011) and Project
"973" (No. G19990
[GenBank]
75600 and No. 200213A711A12). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ These authors contributed equally to this work.
To whom correspondence may be addressed. Tel.: 86-10-65221947; Fax:
86-10-65284074; E-mail:
wzhang{at}pumc.edu.cn.
To whom correspondence may be addressed. Tel.: 86-10-62771493; Fax:
86-10-62773145; E-mail:
raozh{at}xtal.tsinghua.edu.cn.
1 The abbreviations used are: FcR, Fc receptor; KIR, killer cell inhibitory
receptor; LIR, leukocyte Ig-like receptor; r.m.s.d., root mean square
deviation.
2 M. Yang, G. Xu, L. Sun, N. Shi, W. Zeng, W. Zhang, and Z. Rao, unpublished
data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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