Crystal Structure of the Ectodomain of Human Fc{alpha}RI*

Yi Ding {ddagger} § , Gang Xu  ||, Maojun Yang {ddagger}  ||, Min Yao **, George F. Gao {ddagger} §, Linfang Wang ||, Wei Zhang || {ddagger}{ddagger} and Zihe Rao {ddagger} § §§

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Human Fc{alpha}RI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum. Although Fc{alpha}RI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes. The genes of most FcRs are located on chromosome 1 at 1q21 [PDB] –23, whereas Fc{alpha}RI is on chromosome 19, at 19q13.4, 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 (LIRs). The amino acid sequence of Fc{alpha}RI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs. In this work, we analyzed the crystal structure of the ectodomain of Fc{alpha}RI and examined structure similarities between Fc{alpha}RI and KIR2DL1, KIR2DL2 and LIR-1. Our data show that Fc{alpha}RI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and Fc{alpha}RI is evolutionally closer to LIR than KIR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
In humans, IgA is the most abundant immunoglobulin in secretions, and it constitutes about 20% of the immunoglobulin pool in serum (1, 2). Since its turnover rate is faster than other immunoglobulins, the daily production of IgA exceeds all other immunoglobulins combined (3). Undoubtedly, IgA should to play important roles in immune defense against invaded pathogens.

Five types of IgA receptors have been recognized so far. They are Fc{alpha}RI (CD89), the polymeric Ig receptor, Fc{alpha}/µR, the transferrin receptor, and the asialoglycoprotein receptor (1). Among them, Fc{alpha}RI is the only one that specifically binds IgA. On ligation of IgA complexed with antigens, Fc{alpha}RI is able to mediate various cellular responses including phagocytosis, antibody-dependent cell cytotoxicity, oxidative bursts, and release of inflammatory mediators (1).

Fc{alpha}RI 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{alpha}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{alpha}RI are potentially involved in IgA binding (6, 7).

Although Fc{alpha}RI is an immunoglobulin Fc receptor (FcR),1 it differs in many ways with FcRs for other immunoglobulin classes. IgG receptor Fc{gamma}RIII and IgE receptor Fc{epsilon}RI bind antibodies in the near hinge regions and form 1:1 complexes (8, 9), whereas Fc{alpha}RI binds the CH2-CH3 interface of Fc{alpha} (10, 11) and preferably forms 2:1 complex with a single Fc{alpha} homodimer (12). It has been reported that Fc{gamma}Rs and Fc{epsilon}RI use their membrane proximal-domain and linker region binds immunoglobulin (8, 9, 13, 14), whereas Fc{alpha}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 Fc{alpha}RI 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{alpha}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 Fc{alpha}RI expressed in Escherichia coli and its comparison with FcRs, LIR, and KIR.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Protein Expression and Purification—The construction and expression of the extracellular ligand binding domain of a human Fc{alpha}RI will be described in detail elsewhere.2 Briefly, residues 1–207 of the mature sequence were subcloned into a Novagen pET-28a vector using the NcoI and XhoI restriction sites and an E. coli BL21(DE3) strain. Two additional amino acids (Met-Ala) were added to the 5' end of the gene, and a histidine tag (His6) was added to the 3' end to facilitate the expression and purification. The protein was first expressed in an inclusion body form and then reconstituted in vitro. The isolation of the inclusion bodies was started with an intense combined lysozyme/sonification procedure to open virtually all cells. Subsequent washing steps with Triton X-100 and NaCl yielded a product with a purity of >80% as estimated by SDS-PAGE. The inclusion bodies were dissolved in a buffer containing 6 M guanidine hydrochloride and 5 mM dithiothreitol, and incubated for 2 h to unfold completely the misfolded protein of inclusion bodies. Refolding was achieved by dilution of the guanidine-dissolved inclusion bodies dropwise with stirring into the refolding buffer (0.1 M Tris/HCl, 1.5 M guanidine hydrochloride, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.5) at 4 °C. The mixture was stirred for 2–3 days, and then the renatured Fc{alpha}RI was applied to a Q-Sepharose high performance ion-exchange column and further purified on a Superdex-75 column.

Crystallization, Data Collection, and Structure Determination—The Fc{alpha}RI 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 8.0–4.0 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 FoFc map, contoured at 3.5{sigma}, coincided with well defined electron density of a 2FoFc map contoured at 1{sigma}. The N-terminal 2 additional residues (MA), C-terminal 15 residues (DSIHQDYTTQNLILE), and residues 56–59 (FWNE) were disordered in the crystal. The final model contained 191 residues of Fc{alpha}RI and 68 solvent molecules. Rcryst and Rfree were 0.210 and 0.239, respectively, for data in the resolution range 40.0–2.1 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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TABLE I
Data collection, phasing, and refinement statistics

Rmerge = {Sigma}|Ii - <I>|/{Sigma}|I|, where Ii is the intensity of an individual reflection and <I> is the average intensity of that reflection. R-factor = {Sigma}|Fp| -|Fc|/{Sigma}|Fp|, where Fc is the calculated and Fp is the observed structure factor amplitude. Phasing power = Fhcalc/E, where Fhcalc = the heavy atom structure factor amplitude and E = the residual lack of closure error. Rcullis = {Sigma}||Fph ± Fp| -|Fhcalc|/{Sigma}|Fph ± Pp|, where Fph is the derivative structure factor amplitude.

 

For analyses of interdomain angles, contacts, and buried surface areas, D1 was defined as residues 1–100 and D2 was defined as residues 101–195, 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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The crystal structure of the extracellular region of Fc{alpha}RI consists of two Ig-like domains, EC1 (residues Gln1 to Gly100) and EC2 (residues Pro105 to His199) (Fig. 1a). EC1 and EC2 obey the typical heart-shaped arrangement, and a short linker (Leu101 to Lys104) connects them together. Both domains are primarily composed of {beta}-structure arranged into two antiparallel {beta} sheets with a KIR-like folding topology. The sheets are closely packed against each other with the conserved disulfide bridge connecting the strands B and F on the opposing sheets. Three 310 helices are found in N termini of EC1 (Glu2 to Asp4), EF loops of EC1 (Ala71, Asn72, and Lys73) and EC2 (Leu164, Asn165, and Val166), respectively.



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FIG. 1.
Crystal structure of Fc{alpha}RI ectodomain. a, stereo ribbon drawing of the structure of Fc{alpha}RI. EC1 is the N-terminal domain, and EC2 is the C-terminal domain. Disulfide bonds are shown in green. The residues 56–59 and 196–199 were disordered in the electron density map. b, topological diagram of the ectodomain of Fc{alpha}RI. The arrows show the directions of {beta}-strands, whereas the 310 helix structures are represented by two circles. The amino acid residues at each end of {beta}-strands and helices are numbered. c, close-up stereo view of the hydrophobic core in the interdomain interface of Fc{alpha}RI. The 12 residues responsible for stabilizing the hydrophobic core are shown in ball-and-stick representation. Tyr173 (Y173) is colored yellow, Tyr181 (Y181) blue, and Trp183 (W183) green. Other residues are colored using the CPK (Corey-Pauling-Kendrew) convention (blue, nitrogen; red, oxygen; gray, carbon; yellow, sulfur) color scheme.

 

In the EC1 domain, four anti-parallel {beta}-strands (A, B, E, D) oppose a sheet of five {beta}-strands (C',C,F,G,A') (Fig. 1b). There is a {beta}-bulge (Ser91 to Thr93) in the G strand of EC1 splitting the strand into two short {beta}-strands.

The EC2 domain is built up from eight {beta}-strands arranged such that three stands (A, B, E) form one {beta}-sheet and five strands (C', C, F, G, A') form a second {beta}-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 Fc{alpha}RI 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{epsilon}-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 Fc{alpha}RI shows a relatively high degree of homology to the D1 and D2 domains of KIR and LIR, the C{alpha} 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{alpha}RI. In LIR-1 D1, strands C' and D are replaced by two 310 helices. On the other hand, the C' strand of Fc{alpha}RI is shorter than that of KIR2DL1 and KIR2DL2. Hence, the C-C' loop in Fc{alpha}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{alpha}RI. The significance of this is still unknown.



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FIG. 2.
Stereo view of the comparison of Fc{alpha}RI with LIR, KIRs, and FcRs. a, superimpose of Fc{alpha}RI (purple), LIR-1 (orange), and KIR2DL2 (blue). The r.m.s.d. values for superimposition are: 1.44 Å for Fc{alpha}RI EC1 and LIR-1 D1, 1.54 Å for Fc{alpha}RI EC1 and KIR2DL2 D1, 1.34 Å for Fc{alpha}RI EC2 and LIR-1 D2, and 1.18 Å for Fc{alpha}RI EC2 and KIR2DL2 D2. b, comparison of EC1 CC' loop of Fc{alpha}RI with KIR2DL1 and KIR2DL2. The C{alpha} trace of Fc{alpha}RI is colored purple, KIR2DL1 is in green, and KIR2DL2 is in blue. c, comparison of EC1 C to E region of Fc{alpha}RI with other human FcR structures. The C{alpha} traces of Fc{alpha}RI, Fc{epsilon}RI, Fc{gamma}RIIa, Fc{gamma}RIIb, and Fc{gamma}RIII are colored purple, blue, orange, green, and red, respectively.

 

Although LIR-1 does not contain of C' and D strands in the D1 domain, it more closely resembles Fc{alpha}RI 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{alpha} atoms are 1.44 Å for Fc{alpha}RI EC1 and LIR-1 D1, 1.54 Å for Fc{alpha}RI EC1 and KIR2DL2 D1 and 1.77 Å for Fc{alpha}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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FIG. 3.
Comparison of the sequences of human Fc{alpha}RI with human LIR-1, human KIR2DL1, human KIR2DL2, bovine KIR2DL1, and bovine Fc{alpha}IIR. The secondary {beta}-strands of Fc{alpha}RI are shown above the sequence with bold arrows. Conserved residues are shaded. Deletions in the sequences are indicated by dashes.

 

The interdomain angle of Fc{alpha}RI 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{alpha}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{alpha}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{alpha}RI in this region (Fig. 3). Moreover, a bovine IgG2 FcR, Fc{gamma}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{alpha}RI. This Fc{gamma}2R has been previously found to share more similarities to Fc{alpha}RI (41%) than to other types of human Fc{gamma}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{alpha}RI is evolutionally closer to LIR and KIR than to other human FcRs.

As an immunoglobulin Fc receptor, Fc{alpha}RI differs from other FcRs not only in structure but also in its ligand binding characteristics. IgG receptor Fc{gamma}RIII and IgE receptor Fc{epsilon}RI bind antibodies in the near hinge regions and form 1:1 complexes (8, 9). In contrast, Fc{alpha}RI binds the CH2-CH3 interface of Fc{alpha} (10, 11) and preferably forms a 2:1 complex with a single Fc{alpha} homodimer (12). It has been reported that Fc{gamma}Rs and Fc{epsilon}RI use their membrane-proximal domain and linker region to bind immunoglobulin (8, 9, 13, 14), whereas Fc{alpha}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{alpha}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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FIG. 4.
Residues involved in Fc{alpha}RI binding of IgA. a, position of the potential binding area on Fc{alpha}RI. b, molecular surface of Fc{alpha}RI. The residues involved in binding of IgA are highlighted in color. Tyr35 is colored red, Asn44 brown, Arg52 pink, Arg82 blue, Ile83 green, Gly84 dark blue, His85 orange, and Tyr87 purple. The structure was represented by DeepView Swiss-PdbViewer (34).

 

Fc{alpha}RI has six potential N-linked glycosylation sites (Asn44, Asn58, Asn120, Asn156, Asn165, and Asn177). Unglycosylated Fc{alpha}RI has a molecular mass of 30 kDa. When expressed in vivo, its molecular mass is increased to 50–100 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{alpha}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 Fc{alpha}RI 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{alpha}RI binding to IgA.


    FOOTNOTES
 
The atomic coordinates and structure factors (code 1UCT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* 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. Back

These authors contributed equally to this work. Back

{ddagger}{ddagger} 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. Back

2 M. Yang, G. Xu, L. Sun, N. Shi, W. Zeng, W. Zhang, and Z. Rao, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Fei Sun, Feng Xu, and Zhiyong Lou for assistance with data collection at Spring-8 BEAMLINE BL41XU. We also thank Drs. Mark Bartlam and Yiwei Liu for valuable discussion and reading of the manuscript.



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 RESULTS AND DISCUSSION
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