Crystal Structure of Fcγ Receptor I and Its Implication in High Affinity γ-Immunoglobulin Binding*

Background: FcγRI plays important roles in antibody functions. Results: We report the first crystal structure of the extracellular human FcγRI. Conclusion: The receptor D3 domain is positioned away from the IgG binding site, and its shorter D2 domain FG-loop is important for its high affinity. Significance: This work provides insights to the mechanism of FcγRI function and helps to design therapeutic reagents. Fcγ receptors (FcγRs) play critical roles in humoral and cellular immune responses through interactions with the Fc region of immunoglobulin G (IgG). Among them, FcγRI is the only high affinity receptor for IgG and thus is a potential target for immunotherapy. Here we report the first crystal structure of an FcγRI with all three extracellular Ig-like domains (designated as D1, D2, and D3). The structure shows that, first, FcγRI has an acute D1-D2 hinge angle similar to that of FcϵRI but much smaller than those observed in the low affinity Fcγ receptors. Second, the D3 domain of FcγRI is positioned away from the putative IgG binding site on the receptor and is thus unlikely to make direct contacts with Fc. Third, the replacement of FcγRIII FG-loop (171LVGSKNV177) with that of FcγRI (171MGKHRY176) resulted in a 15-fold increase in IgG1 binding affinity, whereas a valine insertion in the FcγRI FG-loop (171MVGKHRY177) abolished the affinity enhancement. Thus, the FcγRI FG-loop with its conserved one-residue deletion is critical to the high affinity IgG binding. The structural results support FcγRI binding to IgG in a similar mode as its low affinity counterparts. Taken together, our study suggests a molecular mechanism for the high affinity IgG recognition by FcγRI and provides a structural basis for understanding its physiological function and its therapeutic implication in treating autoimmune diseases.

the most abundant antibodies in humans and their receptors, which include Fc␥RI(CD64), Fc␥RIIA, B, C (CD32), and Fc␥RIII (CD16) (10), have been extensively characterized. Among them, Fc␥RI, Fc␥RIIA, and Fc␥RIII are activating receptors that either require the association with an immunoreceptor tyrosine-based activation motif (ITAM) containing FcR common ␥ chain (Fc␥RI and Fc␥RIII) or bear an ITAM in their cytoplasmic tail (Fc␥RIIA and IIC) (8). Fc␥RIIB is an inhibitory receptor that contains an immunoreceptor tyrosinebased inhibitory motif within its cytoplasmic domain. According to the binding affinities to their cognate antibodies, Fc␥RI and Fc⑀RI are high affinity receptors with dissociation constants ranging from 10 Ϫ8 to 10 Ϫ10 M (7,8,10), whereas other Fc receptors, such as Fc␥RII and Fc␥RIII, are low affinity receptors with dissociation constants approximately ϳ10 Ϫ5 -10 Ϫ7 M. Structurally, Fc␥RI is the only Fc receptor with three extracellular Ig-like domains (designated herein D1, D2, and D3), whereas all other Ig superfamily FcRs contain two Ig-like domains. To date, several structures of low affinity Fc␥R and their complexes with IgG-Fc have been published (11,12). However, no atomic structural information is available for Fc␥RI, and consequently, the mechanism of high affinity receptor-antibody recognition remains to be elucidated.
The interaction between Fc␥Rs and IgGs also holds profound implications for the development of therapeutic treatments for antibody-mediated autoimmune diseases. For example, the infusion of high doses of serum IgG (intravenous immunoglobulins) has been used to treat immune thrombocytopenia and Kawasaki disease in human and arthritis as well as nephrotoxic nephritis in mouse models (13)(14)(15)(16)(17). Because of its unique high affinity antibody binding, Fc␥RI has been developed as a potential therapeutic reagent to treat immune complex-mediated disease (18,19). To further understand the high affinity antibody recognition by Fc␥RI and to facilitate the development of Fc␥RI-mediated immunotherapy, we determined the structure of the full-length extracellular domain of human Fc␥RI, investigated the molecular basis of its high affinity IgG binding using site-directed mutagenesis, and assessed the effects of IgG sialylation on Fc␥RI binding. Our study provides structural insights for understanding the physiological function of the high affinity receptor for IgG.

EXPERIMENTAL PROCEDURES
Protein Expression, Purification, and Crystallization-The ectodomain of Fc␥RI (residues 1-282) with its native signal sequence was expressed in Chinese hamster ovary DXB-11 cells and purified via IgG affinity chromatography followed by cation exchange and gel filtration chromatography as described previously (18,19). Before crystallization, Fc␥RI was dialyzed against 10 mM Hepes (pH 7.4) and 0.1 M NaCl and concentrated to a final A 280 nm of 29. The crystals used for data collection were grown in hanging drops at 22°C using 1 l of protein and 1 l of reservoir solution (10% PEG8000 and 10% PEG1000).
Diffraction Data Collection and Structure Determination-The crystals were immersed in cryoprotectant (10% PEG8000, 10% PEG1000, and 20% glycerol) before flash-cooling in liquid nitrogen. The x-ray data were collected to 2.65 Å resolution at SER-CAT beamlines and processed with HKL2000 (20) (see Table 1). Fc␥RI crystals belong to space group P3 2 21 with the cell constants of a ϭ b ϭ 92.83 Å and c ϭ 90.76 Å. The structure of the Fc␥RI ectodomain was solved by a molecular replacement method with the program Bables (21) and Phaser (22) in CCP4 packages (23). More specifically, the Ig domains 1 and 2 (D1 and D2) of Fc␥RI were solved by Bables, which picked the structure of human high affinity IgE receptor (PDB ID 1F2Q) as the best search model. Then with the D1 and D2 domains fixed in position, the D3 domain of Fc␥RI was solved by Phaser using the D2 domain of Fc␥RIIA (PDB ID 3D5O) as the search model. Model building and refinement were carried out using Coot (24) and autoBuster (25). The overall electron density of the receptor is of good quality (supplemental Fig. S1). Carbohydrate molecules were added manually using (2F o Ϫ F c ) electron density maps contoured at 1.0 (standard deviation of the map) and refined. The residues are numbered consistent with FCGR1_HUMAN in the Swiss-Prot entry. The final model includes residues 21-282 of the Fc␥RI ectodomain, with the exception of one loop region, residues 218 -223. The Ramachandran statistics were generated with PRO-CHECK (26). Hinge angles were calculated using program HINGE (27), and the buried surface area was calculated using AreaIMol (28) in CCP4 packages. All structure figures were generated with PyMOL (29). The coordinates of Fc␥RI have been deposited in the Protein Data Bank under the ID code 3RJD.
Site-directed Mutagenesis of Fc␥RIII-The recombinant Fc␥RIII with mutated FG loop in the D2 domain were generated by site-directed mutagenesis. Briefly, the FG loop ( 171 LVGSKNV 177 ) of Fc␥RIII that was previously cloned in    Fc␥RIII proteins were expressed and purified as described previously (30).
Surface Plasmon Resonance Solution Binding Experiments-Surface plasmon resonance measurements were performed using a BIAcore 3000 instrument and analyzed with BIAevaluation 4.1 software (Biacore AB). Different human IgG subclasses were obtained from Athens Research and Technology (Athens, GA). The sialylated and asialylated Fc samples were gifts from Dr. Jeffrey Ravetch (Rockefeller University). To measure the affinity to different subclasses of human IgGs, Fc␥RI was immobilized on carboxylated dextran CM5 chips (Biacore AB) to 200 -500 response units using a primary aminecoupling in 10 mM sodium acetate (pH 4.0). The analytes consisted of serial dilutions of IgG subclasses or sialylated Fc between 3.72 M and 29 nM in a buffer containing 10 mM Hepes (pH 7.4) and 0.15 M NaCl. To measure the binding to Fc␥RIII wild type and mutant proteins, human IgG 1 was immobilized on CM5 chip to 20 -150 response units as described above. The analytes consisted of serial dilutions of Fc␥RI and Fc␥RIII wild type and mutant proteins between 7.89 and 0.03 M in a buffer containing 10 mM Hepes (pH 7.4) and 0.15 M NaCl plus 0.01% P20 (Biacore AB). The dissociation constants were obtained by kinetic curve-fitting for the binding of Fc␥RI to IgGs and steady-state fitting for the binding of Fc␥RIII and its mutants to IgG 1 , respectively, using BIAevaluation 4.1 (BIAcore Inc.).

RESULTS AND DISCUSSION
The Overall Structure of the Fc␥RI Ectodomain-The crystal structure of the extracellular domain of Fc␥RI was determined to 2.65 Å by molecular replacement (Table 1). Carbohydrates were observed and modeled at six glycosylation sites, including asparagine residues 59, 78, 152, 159, 163, and 195 (Fig. 1A). Fc␥RI is the only Fc receptor that contains three Ig domains, and the overall shape of Fc␥RI resembles the shape of a sea horse, with the interdomain hinge angles calculated to be 35°b etween the D1 and D2 and 120°between the D2 and D3 domains. All three domains display a C2-set Ig fold consisting of two antiparallel sheets formed by strands ABE and CFG. The pairwise structural superimposition between the three Ig domains resulted in root mean square deviations of ϳ1.7 Å.
Structure Comparison with Other Fc Receptors-The structures of the low affinity Fc␥RII and Fc␥RIII can be well superimposed onto each other with root mean square deviations of 0.9 -1.1 Å (30 -33). The hinge angles between the D1 and D2 domains of low affinity Fc␥Rs are remarkably similar, ranging from 55°to 60°. Fc␥RI, in contrast, displays a smaller D1-D2 domain hinge angle of 35°than those of Fc␥RII and Fc␥RIII but similar to the 39°hinge angle of Fc⑀RI ( Fig. 2A) (34). This hinge angle is stabilized by extensive interactions between the D1 and D2 domains, including two interdomain salt bridges, three hydrogen bonds, and numerous hydrophobic interactions ( Fig.  1B and Table 2). Some of the Fc␥RI hinge core residues, such as Phe-34, Glu-37, and Leu-106 in the D1-D2 hinge region are conserved with Fc⑀RI but not with Fc␥RII or RIII (Fig. 2B), further supporting their structural similarity. The hinge core in Fc␥RII and III, in contrast, contains one salt bridge and one hydrogen bond. Except for three conservative amino acid changes, Val-33 to Ile, Glu-99 to Gln, and Val-109 to Ala, the rest of the 15 residues in the D1-D2 hinge core are invariant between human and murine Fc␥RI (Fig. 2B). The D2-D3 hinge is unique to the three domains of Fc␥RI. It consists of one salt bridge between Glu-116 from the D2 domain and Lys-271 of the D3 domain and four hydrogen bonds involving Gly-117, Lys-157, Glu-187, and Leu-188 from the D2 domain and Lys-271 and Asn-268 from the D3 domain (Fig. 1C). Except for a conservative Asn-268 to Ser replacement, the other six residues involved in the D2-D3 hinge core are invariant between human and murine Fc␥RI. Despite fewer interdomain contacts and the appendage nature of the D3 domain, the entire domain has well defined electron densities and does not appear to exist in multiple conformations in the crystal. A plot of the crystallographic B-values for all C␣ atoms of Fc␥RI shows similar thermomobility indices for the D2 and D3 domains (supplemental Fig. S1), further supporting a non-flexible conformation for the D3 domain of the receptor. The overall conservation in both the D1-D2 and the D2-D3 hinge cores between human and murine Fc␥RI suggests that the murine Fc␥RI adopts a similar structure to that of the human Fc␥RI.
The Role of D3 Domain in Fc␥RI Recognition of IgG-Previous mutational studies suggested the importance of the receptor D2 and D3 domains to the high affinity IgG binding (35,36). The structures of Fc␥RIII and Fc⑀RI in complex with their Fc showed a similar ligand binding mode involving the receptor BC, CЈE, FG loops, and the CЈ strand on the D2 domain despite the differences in their structures and binding affinities (11,12,37). Several of the Fc contacting residues of Fc␥RIII and Fc⑀RI, including Trp-104 and Trp-127 that form a sandwich around Pro-329 of Fc, are conserved in the high affinity Fc␥RI structure (Fig. 2, supplemental Fig. S2), arguing a similar Fc recognition mode for Fc␥RI. When the structure of Fc␥RI is superimposed onto that of the Fc␥RIII-Fc complex, its D3 domain is positioned ϳ30 Å away from the putative IgG-Fc interface, suggesting that the D3 domain of Fc␥RI is unlikely to contact directly with IgG-Fc (Fig. 3A). However, the D3 domain of Fc␥RI may indirectly affect IgG binding through either stabilizing the con- formation of the receptor or making the receptor protruding more into the extracellular space than its two-domain Fc receptor counterparts and, thus, more available for ligand binding.
The Mechanism of High Affinity Fc␥RI Recognition of IgG-Although Fc␥RIII and Fc⑀RI recognize Fc in similar binding modes, the structures of their ligand complexes show a slightly more buried interface area in Fc⑀RI/IgE-Fc (1883 Å 2 ) than in Fc␥RIII/IgG-Fc (1798 Å 2 ) (11,37). It remains to be seen if Fc␥RI makes more extensive IgG contacts than the low affinity Fc␥Rs. The known structures also showed that the D2 domain FG-loop on both Fc⑀RI and Fc␥RIII formed critical interface contacts with Fc, and the exchange of the D2 domain FG-loop of Fc⑀RI to Fc␥RII confers detectable IgE binding (38). Interestingly, the D2 domain FG-loop of Fc␥RI ( 171 MGKHRY 176 ) contains a single-residue deletion compared with Fc␥RII/Fc␥RIII and Fc⑀RI (Fig. 2B). This deletion is not present on the D1 domain FGloop of Fc␥RI and is conserved in all species from human to mouse, indicating a potential functional relevance of this unique shorter FG-loop in Fc␥RI binding to IgG. Simple structural modeling to replace the longer FG-loop residues of Fc␥RIII (LVGSKNV) with those of corresponding Fc␥RI in the Fc␥RIII/IgG 1 -Fc structure showed that instead of making more potential contacts with IgG-Fc, the bulkier Fc␥RI residues were in steric clash with IgG 1 -Fc. Similarly, when the structure of Fc⑀RI was superimposed onto that of Fc␥RIII-Fc complex, it revealed a steric clash between the bulkier Fc⑀RI FG-loop residues and IgG-Fc (Fig. 3B), suggesting a potential conformational conflict between IgG-Fc and the longer FG-loop of Fc⑀RI. Indeed, structural overlay showed an ϳ3 Å displacement in the position of the shorter Fc␥RI FG-loop relative to that of Fc␥RIII (Fig. 3B). To further investigate the contribution of the Fc␥RI FG-loop and its one-residue deletion to the receptor-IgG binding affinity, we mutationally replaced the FG-loop of Fc␥RIII ( 171 LVGSKNV 177 ) with both the wild type Fc␥RI loop ( 171 MGKHRY 176 ) and a mutant Fc␥RI loop, which contains a Fc␥RIII-like valine insertion at residue 172 ( 171 MVGKHRY 177 ). The wild type Fc␥RI and Fc␥RIII bound to immobilized IgG 1 with affinities of 0.02 and 1.5 M, respectively (Table 3, supplemental Fig. S3A). When the recombinant mutant Fc␥RIII carrying a shorter, wild type Fc␥RI FG-loop ( 171 MGKHRY 176 ) was examined for IgG 1 binding, it resulted in an affinity of 0.1 M, 5-fold lower than that of wild type Fc␥RI but 15-fold higher than that of the wild type Fc␥RIII (    (11,12,42). This, however, does not explain the specificity of Fc␥RI, which binds IgG 4 but not IgG 2 in similar affinity as IgG 1 (Table 3, supplemental Fig. S3B). The current structure of Fc␥RI and its modeled complex with Fc provide a possible explanation to this isotype-dependent IgG 4 binding specificity (supplemental Fig. S2). This isotype specificity difference between Fc␥RI and Fc␥RIII may be attributed, based on the modeling of Fc␥RI-IgG interactions, to a hydrogen bond between Tyr-176 in the FG-loop of Fc␥RI but not Val in Fc␥RIII and the conserved lower hinge residues of IgG 1,3 and IgG 4 but not IgG 2 .
Effect of Sialylation on Fc Binding by Fc␥RI-On both chains of Fc, ordered carbohydrate moieties are attached to one single conserved glycosylation site, Asn-297. Unlike most glycosylations, these carbohydrates on Fc are extensively interacting with the inner face of the C H 2 domain to provide conformational stability for the Fc region (11,12). Indeed, removal of these carbohydrates by enzymatic digestion dramatically decreased the binding affinity of IgGs to their receptors (43). Structural studies have shown that deglycosylated Fc undergoes remarkable conformational changes in the C H 2 domain, although C H 3 domains are intact (44). Even though these carbohydrate moieties are away from the receptor contact area, they are known to affect Fc receptor binding. Recently, sialylated Fc was proposed to harbor the anti-inflammatory activity of intravenous immunoglobulins as sialic acid-enriched IgG showed a 10-fold affinity reduction in binding to the low affinity Fc␥RIIB and Fc␥RIII (45). To investigate whether sialylation also affects the binding of Fc to Fc␥RI, we measured the receptor binding to a sialic acid-enriched Fc fragment using surface plasmon resonance analysis (Table 3, supplemental Fig. S3C). The result showed that Fc␥RI bound to the sialylated Fc with a modest 2-fold reduction in affinity (83 nM) compared with native Fc (42 nM). It remains to be seen whether the reduced binding affinity of Fc␥RI to sialylated Fc has any physiological relevance in the immunosuppressive activity of intravenous immunoglobulins.