The Structure of a Human Type III Fcgamma Receptor in Complex with Fc*

Sergei RadaevDagger , Shawn Motyka§, Wolf-Herman Fridman, Catherine Sautes-Fridman, and Peter D. SunDagger ||

From the Dagger  Structural Biology Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852, the § Biochemistry, Cellular, and Molecular Biology Program, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, and the  Laboratory of Cellular and Clinical Immunology, INSERM Unit 255, Curie Institute, 75005 Paris, France

Received for publication, January 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Fcgamma receptors mediate antibody-dependent inflammatory responses and cytotoxicity as well as certain autoimmune dysfunctions. Here we report the crystal structure of a human Fc receptor (Fcgamma RIIIB) in complex with an Fc fragment of human IgG1 determined from orthorhombic and hexagonal crystal forms at 3.0- and 3.5-Å resolution, respectively. The refined structures from the two crystal forms are nearly identical with no significant discrepancies between the coordinates. Regions of the C-terminal domain of Fcgamma RIII, including the BC, C'E, FG loops, and the C' beta -strand, bind asymmetrically to the lower hinge region, residues Leu234-Pro238, of both Fc chains creating a 1:1 receptor-ligand stoichiometry. Minor conformational changes are observed in both the receptor and Fc upon complex formation. Hydrophobic residues, hydrogen bonds, and salt bridges are distributed throughout the receptor·Fc interface. Sequence comparisons of the receptor-ligand interface residues suggest a conserved binding mode common to all members of immunoglobulin-like Fc receptors. Structural comparison between Fcgamma RIII·Fc and Fcepsilon RI·Fc complexes highlights the differences in ligand recognition between the high and low affinity receptors. Although not in direct contact with the receptor, the carbohydrate attached to the conserved glycosylation residue Asn297 on Fc may stabilize the conformation of the receptor-binding epitope on Fc. An antibody-Fcgamma RIII model suggests two possible ligand-induced receptor aggregations.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Fc receptors, which are expressed on the majority of hematopoietic 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. The three major types of Fc receptors are Fcgamma , Fcepsilon , and neonatal Fc receptors. Except for the neonatal Fc receptor and Fcepsilon RII (CD23), which are related structurally to class I major histocompatibility antigens and C-type lectins, respectively, all other known Fc receptors are members of the immunoglobulin superfamily (1, 2). Among them, Fcgamma RI and Fcepsilon RI1 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 Fcgamma RII and Fcgamma RIII, are low affinity receptors with dissociation constants ranging from 10-5 to 10-7 M (3-5). In addition to variations in affinity, each receptor displays distinct IgG subtype specificities. Unlike the high affinity receptors that can bind monomeric antibodies, the low affinity receptors preferentially bind to and are activated by immune complexes.

Human Fcgamma RIII exists as two isoforms, Fcgamma RIIIA and Fcgamma RIIIB, that share 96% sequence identity in their extracellular immunoglobulin-binding regions. Fcgamma RIIIA is expressed on macrophages, mast cells, and natural killer cells as a transmembrane receptor. In contrast, Fcgamma RIIIB, present exclusively on neutrophils, is anchored by a glycosyl-phosphatidylinositol linker to the plasma membrane. Although Fcgamma RIIIA associates with the immunoreceptor tyrosine-based activation motif containing Fcepsilon RI gamma -chain or the T cell receptor zeta -chain for its signaling, Fcgamma RIIIB lacks a signaling component. Nevertheless, it plays an active role in triggering Ca2+ mobilization and in neutrophil degranulation (6, 7). In addition, Fcgamma RIIIB, in conjunction with Fcgamma RIIA, activates phagocytosis, degranulation, and the oxidative burst that leads to the clearance of opsonized pathogens by neutrophils. A soluble form of Fcgamma RIIIB was reported to activate the CR3 complement receptor-dependent inflammatory process (8).

The Fc binding region on Fcgamma RII and Fcgamma RIII has been identified through the work of chimeric receptors with Fcepsilon RI 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 (9-11). Similar regions on the alpha -chain of Fcepsilon RI were also identified to be critical for IgE binding affinity (12). The receptor binding site on Fc has been located through the construction of chimeric IgG molecules and mutational analysis at the lower hinge region, residues located in the hinge region between the CH1 and CH2 domains and immediately adjacent to the N terminus of the CH2 domain of IgG (13-15). 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 Fcepsilon RI binding (16). Apart from the lower hinge region, a few residues on the CH2 domain of an IgG2b were also suggested to interact with the receptor (17). However, with the exception of the neonatal Fc receptor, the molecular recognition between the Fc receptors and Fc remains to be elucidated (18).

The recent crystal structures of Fcepsilon RIalpha , Fcgamma RIIA, and Fcgamma RIIB have each revealed a conserved Ig-like structure, with particularly the small hinge angle between the two Ig-like domains, which is unique to the Fc receptors (19-21). We report here the crystal structure of a human Fcgamma RIII in complex with the Fc portion of a human IgG1 determined from two crystal forms.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Protein Expression, Purification, and Crystallization-- The extracellular part of the human Fcgamma RIIIb receptor, residues 1-172, was expressed as Escherichia coli inclusion bodies and then reconstituted in vitro as described previously (22). Fc fragments of human IgG1 antibody were prepared by the papain digestion as previously reported (23, 24).

The complex of Fc and Fcgamma RIII was prepared by mixing both components in a 1:1 molar ratio and concentrating to 8-15 mg/ml for crystallization. Single crystals of orthorhombic and hexagonal forms were obtained by vapor diffusion in hanging drops at room temperature under slightly different crystallization conditions. Rod-shaped crystals of the orthorhombic form were grown from 10% polyethylene glycol 4000 and 50 mM Hepes at pH 6.5. They appeared after 2-3 days and grew to an average size of 0.05 × 0.05 × 0.2 mm in ~2 weeks. Crystals of the second form, hexagonal bipyramids, were crystallized from 5% polyethylene glycol 6000 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 1 month.

Structure Determination-- After briefly 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 charge-coupled device detector at the X9B beam line of the National Synchrotron Light Source at the Brookhaven National Laboratory and processed with HKL2000 (25). The hexagonal crystals belong to space group P6522 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 P212121 with cell dimensions of a = 76.4, b = 102.8, and c = 123.3 Å diffracted to 3.0 Å. Both crystal forms contain one molecule of Fcgamma RIII and one molecule of Fc in the asymmetric unit.

The structure of the Fcgamma RIII·Fc complex in both crystal forms was determined by molecular replacement. Polyalanine models of Fc (PDB accession number 1FC1) and Fcgamma RIII were used in rotation and translation searches using 15-4 Å data for both crystal forms. An I/sigma (I) cutoff of 2.0 was used for hexagonal data during search procedures. The position of the Fc molecule was determined in both crystal forms using AmoRe (26). 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 (RF) of 51% (CC = 51% and RF = 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 RF = 52% (CC = 46% and RF = 50% after rigid-body refinement in AmoRe). The position of Fcgamma RIII was determined in both crystal forms using the program EPMR (27) with a polyalanine model of Fcgamma RIII and the position of the Fc molecule fixed. Clear solutions were obtained for both crystal forms with CC = 56% and RF = 48% for orthorhombic crystal and CC = 55% and RF = 48% for hexagonal crystal, respectively. After rigid-body refinement of individual domains of the Fcgamma RIII·Fc complex modeled as polyalanine using CNS (28) 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 version 0.9. Model adjustments and rebuilding were done using the program O (29). Carbohydrate molecules were added manually using 2Fo - Fc electron density maps contoured at 1.0sigma and refined. The final model includes residues 5-172 of Fcgamma RIII, residues 235-444 for one chain of Fc, and residues 233-443 for the other chain of Fc.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Overall Structure of the Complex-- Crystals of a human Fcgamma RIII receptor in complex with a human Fc fragment of IgG1 were grown in two forms under different conditions. The orthorhombic crystals belong to the space group of P212121 and diffract to 3.0-Å resolution, whereas the hexagonal crystals have P6522 space group symmetry and diffract to 3.5-Å 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 Rcryst = 23.0% and Rfree = 28.9% for the orthorhombic form and Rcryst = 24.9% and Rfree = 32.6% for the hexagonal form, respectively (Table I). The electron density is continuous throughout the complex in the final 2Fo - Fc map except for three surface loops of Fcgamma RIII (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 crystals contain one Fcgamma RIII and one Fc molecule in each asymmetric unit, suggesting a 1:1 stoichiometry for the binding between the receptor and Fc (Fig. 1). This is consistent with earlier binding studies using non-equilibrium and equilibrium gel filtration experiments (22). The conformation of the Fcgamma RIII·Fc complex is essentially identical in both crystal forms, including the conformation of the visible carbohydrate moieties on the Fc fragment (Fig. 2A).

                              
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Table I
Data collection and refinement statistics


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Fig. 1.   Ribbon drawing showing front view (A) and side view (B) of the Fcgamma RIII·Fc. The side view of the complex is rotated approx 90° from the front view. Fcgamma RIII is shown in green, Fc is cyan. Positions of D1 and D2 domains of Fcgamma RIII as well as CH2 and CH3 domains of Fc are marked. Carbohydrate moieties are shown in gray.


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Fig. 2.   A, superposition of an alpha -carbon trace of the Fcgamma RIII·Fc complex determined from both the orthorhombic (green and cyan for Fcgamma RIII and Fc, respectively) and hexagonal (orange and red) crystal forms. B, superposition of the structure of Fcgamma RIII in the receptor·Fc complex (green) with that of ligand free receptor (orange). C, superposition of the structure of Fc in Fcgamma RIII·Fc complex (cyan) with that of unbound Fc (red).

The Structure of Fcgamma RIII-- The structure of Fcgamma RIII 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 Calpha atoms (Fig. 2B) (22). The hinge angle between the N-terminal (D1) and the C-terminal (D2) domains is 60°, which is slightly larger than the 50° value observed in the ligand free receptor. However, no significant change in the receptor conformation is observed upon complex formation (Fig. 2B).

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, CH2 and CH3. The overall shape of the Fc fragment resembles that of a horseshoe with the two CH3 domains packing tightly against each other at the bottom of the horseshoe and the CH2 domains held apart by carbohydrate moieties attached to the glycosylation site Asn297 from both chains forming the opening of the horseshoe. Well defined electron density throughout the Fc allowed for unambiguous tracing of residues Leu234 to Ser444 in chain A and Pro232 to Leu443 in chain B of Fc, including the lower hinge regions, Leu234-Pro238. The structure of the Fc fragment in complex with Fcgamma RIII does not differ significantly from that observed in the structures of an unbound Fc fragment and a murine intact IgG2a antibody (30, 31) (Fig. 2C). However, the 2-fold symmetry relating the two chains of Fc in other unligated Fc structures, is no longer retained in the structure of the complex. The horseshoe-shaped Fc is slightly more open at the N-terminal end of the CH2 domains in the Fcgamma RIII·Fc structure compared with other known structures of Fc. The hinge angle between CH2 and CH3 domains of chain A (Fc-A) is 95° and 100° in the orthorhombic and the hexagonal crystals, respectively, ~10° larger than the corresponding angle of chain B (Fc-B) and the 84°-89° angle observed in all structures of ligand-free Fc (Fig. 2C).

The Interface between Fcgamma RIII and Fc-- The receptor binds to Fc at the center of the horseshoe 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) (Fig. 1). Such 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, resulting in a 1:1 stoichiometry for the receptor·Fc recognition. The structural implications of the activation of Fc receptors is profound. Particularly, the 1:1 receptor·Fc binding stoichiometry highlights the importance of antigen in the receptor aggregation. In contrast to the high affinity Fcgamma RI and Fcepsilon RI receptors, the binding of immunoglobulins to Fcgamma RIII in the absence of antigen does not lead to receptor aggregation. It can be argued that a 1:1 receptor-ligand stoichiometry ensures the need for antigens in forming the receptor aggregation by eliminating the possibility of Fc-mediated receptor aggregation as suggested in a 2:1 stoichiometry. Precluding receptor aggregation mediated by Fc alone also eliminates the potential deleterious effect of antibodies whose concentration in vivo are often much higher than that of antigen.

The receptor·Fc complex buries ~1453 Å2 of solvent-accessible area (Fig. 3A). The interface between Fcgamma RIII and Fc molecules shows poor shape complementarity with a mean shape correlation statistic of 0.53 (32), less than those between T-cell antigen receptor and Class I major histocompatibility complex 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 (Fig. 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' beta -strand. The BC loop is positioned across the horseshoe opening making contact with 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 Fcgamma RIII protrudes into the opening between the two chains of Fc (Fig. 1A). All three receptor loops (BC, C'E, and FG) were implicated in Fc binding through earlier studies of chimeric Fcgamma RII/Fcepsilon RI receptors and through site-directed mutagenesis (9, 10, 12). On the Fc side of the complex, interactions with the receptor are dominated by residues Leu234-Pro238 of the lower hinge (Table II), consistent with results form earlier mutational studies (2). In particular, Hinge-A and -B together contribute ~60% of the overall receptor·Fc interface area (Fig. 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 (30, 33-35). In contrast, residues of both Hinge-A and -B are clearly visible in the electron density maps from both crystal forms, suggesting that the binding of Fcgamma RIII stabilizes the lower hinge conformation of Fc.


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Fig. 3.   Fcgamma RIII·Fc interface. A, surface representation of Fcgamma RIII. The interface region is color-coded. Regions involved in the interactions with Hinge-A and Hinge-B are yellow and green, respectively. All other contact areas are colored in blue. Lower hinge regions of Fc are in ball-and-stick representation (red). B, interactions between Fcgamma RIII (green) and chain A of Fc (cyan). The glycosylation residue, Asn297, is also shown. C, interactions between Fcgamma RIII (green) and chain B of Fc (cyan). The side chain of Val158 is omitted for clarity. There is a hydrogen bond between carbonyl group of Val158 and backbone nitrogen of Gly236 (Table II) 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' beta -strands of Fcgamma RIII, which play an important role in the interactions, are labeled. Some secondary structure elements of Fc lying behind and not contributing to the binding shown as semi-transparent. Carbohydrate moieties have been omitted for clarity.

                              
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Table II
Interface contacts between Fcgamma RIII and Fc

A combination of salt bridges, hydrogen bonds, and hydrophobic interactions contributes to the receptor·Fc recognition. Specifically, the interface between Fcgamma RIII and Fc-A is dominated by hydrogen bonding interactions, whereas the hydrophobic interactions occur primarily at the interface between Fcgamma RIII 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 (Fig. 3, B and C, and Table II). Seven hydrogen bonds are distributed across the receptor and Fc-A interface and two are at the receptor and Fc-B interface. Alanine mutations, such as the H134A mutant of Fcgamma RII 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 (10). A hydrophobic core is formed between Trp90, Trp113 of the receptor, and Pro329 of the CH2 domain of Fc-B (Fig. 3C). This hydrophobic core extends further to include Val158, the aliphatic side chain of Lys161 of the receptor and Leu235 of Hinge-B. Mutations of both Trp113 and Lys161 in Fcgamma RIII lead to the loss in receptor function (11, 36). The side chain of Leu235 on the Fc-B packs tightly against Gly159 of the receptor leaving little space to accommodate any residues larger than Gly at this position. A G159A mutation on chimeric Fcgamma RII resulted in the complete disruption of Fc binding, presumably due to the steric hindrance between Leu235 and the beta -carbon of the alanine mutant at position 159 (9). Of particular interest is Trp113 of the receptor, which when mutated to Phe resulted in the loss of Fc binding. This residue is not only 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 Fcgamma RIII. A W113F mutation would result in the loss of this wedge and lead to a disruption in binding by altering the orientation between the D1 and D2 domains.

Comparison of the Structures of Receptor·Fc Complexes-- Including the two crystal forms described in this work, there are a total of four Fc receptor and Fc complex structures available to date. A comparison among these structures reveals the conformation flexibility of this receptor·ligand complex and helps to explain the molecular interactions that differentiate the high from the low affinity receptors.

The two crystal forms of Fcgamma RIII·Fc complexes determined in the present study are essentially identical and can be readily superimposed with a root mean square (r.m.s.) deviations of 1.1 Å among all Calpha atoms. The superposition of the hexagonal form onto the published Fcgamma RIII·Fc complex resulted in an r.m.s deviation of 0.5 Å for all Calpha atoms (37) (Fig. 4A). An analysis of the interdomain hinge angles shows that the CH2-CH3 hinge angle is 10° larger in the structure of the Fcgamma RIII·Fc complex than it is in the structure of an intact IgG2a antibody (35) or the structures of ligand-free Fc (30) (Table III). This results in a slightly more open conformation of the Fc when ligated to the receptor. Apart from the small change of the hinge angle, neither the Fc nor the receptor displays significant conformational change upon complex formation. The agreement between the orthorhombic and hexagonal crystal forms of the complex indicates a well defined receptor-ligand recognition free from conformational flexibility.


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Fig. 4.   Superposition of the Fcgamma RIII·Fc complex determined from the orthorhombic crystal form onto the structures of previously determined complexes Fcgamma RIII·Fc (46) and Fcepsilon RI·Fc (45). A, superposition of the Fcgamma RIII·Fc complex determined from the orthorhombic crystal form (green and cyan for Fcgamma RIII and Fc, respectively) and previously determined structure of Fcgamma RIII·Fc complex (46) (orange and red for Fcgamma RIII and Fc, respectively). B, superposition of the structure of Fcgamma RIII·Fc (green and cyan for Fcgamma RIII and Fc, respectively) and Fcepsilon RI·Fc (orange and red for Fcepsilon RI and Fc, respectively) complex (45). C, a definition of the hinge angles. D, an enlarged view of superposition of Fcgamma RIII·Fc (green and cyan) and Fcepsilon RI·Fc (red and orange) complexes in the interface area. The lower hinge regions are labeled. The view is identical to that in B.

                              
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Table III
Hinge angle comparison of free and receptor complexed Fc
The hinge angles are defined in Fig. 4C.

The comparison between the structure of the Fcgamma RIII·Fc complex and that of the Fcepsilon RI·Fc complex has provided further insight into the molecular basis of the receptor affinity (38). Overall, a similar mode of receptor-ligand recognition was observed in both the Fcgamma RIII·Fc and the Fcepsilon RI·Fc complexes with an r.m.s. deviation of 1.5 Å between all the Calpha atoms. In fact, most of the structural difference resulted from the small variation between the CH2-CH3 and Cepsilon 3-Cepsilon 4 interdomain hinge angles (Fig. 4, B and C). This angle is ~10° smaller in the Fcepsilon RI·Fc complex structure, resulting in a slightly closed conformation of Fc compared with that of the Fcgamma RIII·Fc complex.

Detailed structural analysis shows that the interface area buried in the high affinity Fcepsilon RI·Fc complex (1850 Å2) is 400 Å2 more than that in the low affinity Fcgamma RIII·Fc complex (1453 Å2). This is primarily due to a more extensive interaction observed between the receptor and the non-lower hinge residues of Fc in the high affinity complex than in the low affinity receptor complex. Of the total interface area of the Fc, the lower hinge and non-lower hinge regions contribute 870 and 580 Å2, respectively, in the Fcgamma RIII·Fc structure. The corresponding regions contribute 740 and 1110 Å2, respectively, in the Fcepsilon RI·Fc structure. This results in approximately twice as much interface area contributed by non-lower hinge residues in the high affinity receptor·ligand complex than in the low affinity receptor·ligand complex. Structurally, the lower hinge of IgE-Fc adopts a very different conformation than that of IgG-Fc in their respective receptor complexes (Fig. 4D). This conformation difference may enable the high affinity Fcepsilon RI to interact more extensively with its ligand.

Although the overall pattern of the receptor·Fc interactions, namely a preference for hydrogen bonding in the Fc A-chain part of the interface and hydrophobic contacts in the Fc B-chain part of the interface are preserved in both the Fcgamma RIII·Fc and Fcepsilon RI·Fc complexes, significant differences were also observed. First, there are more extensive hydrophobic interactions between Fcepsilon RI and IgE-Fc than those between Fcgamma RIII and IgG-Fc. Although the tryptophan-proline sandwich formed by Trp90, Trp113 of Fcgamma RIII and Pro329 of Fc-B (the corresponding Trp87, Trp110, and Pro426 residues in Fcepsilon RI·Fc) is preserved in both structures, additional bulky residues, such as Trp130, found in both Fcepsilon RI and IgE-Fc may also contribute to stronger hydrophobic interactions in the Fcepsilon RI·Fc complex compared with that of the Fcgamma RIII·Fc complex. Second, a more extensive network of hydrogen bonds and salt bridges exists at the Fcepsilon RI·Fc interface compared with that of Fcgamma RIII·Fc. Furthermore, the hydrogen bonds in the Fcepsilon RI·Fc interface are formed mostly between the side-chain atoms whereas those in the Fcgamma RIII·Fc interface are formed primarily between the main-chain atoms or between the main-chain and side-chain atoms. There are two salt bridges Lys117-Asp362 and Glu132-Arg334 observed between Fcepsilon RI and Fc but only one, Lys120-Asp265, is conserved in the low affinity complex between Fcgamma RIII and Fc.

Our results suggest that multiple interactions contribute to the observed receptor-ligand affinity difference and that the higher affinity recognition includes more extensive hydrophobic interface area as well as more prominent electrostatic interactions.

Conserved Receptor·Fc Binding Interface-- Of the 13 receptor interface residues, four (Trp90, Trp113, Lys131, and Gly159) are invariant among all human Fcgamma receptors (Fig. 5A). Three of them are also conserved in the alpha -chain of Fcepsilon RI. Gly159 in Fcgamma receptors is replaced with Trp in Fcepsilon RI. Because Gly159 is in close contact with Leu235 from the lower hinge of Fc-B, replacement of this residue with Trp may result in the observed difference of the lower hinge conformation in IgE. Three other interface residues, Lys120, Tyr132, and Val158, 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 Lys120 and Asp265 of the Fc appears to be absent in Fcgamma RI but conserved in all other Fcgamma receptors and in Fcepsilon RI. The other six interface residues, Ile88, Asp129, His134, His135, Arg155, and Lys161 are less well conserved among the receptors. Of these, variation at His134 and His135 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 particular interest is the comparison between the interface residues of Fcgamma RI and those of Fcgamma RIII. The binding affinity of Fcgamma RIII is at least 100-fold weaker than that of Fcgamma RI. Among the receptor·Fc interface residues, only four are different between Fcgamma RIII and Fcgamma RI. These are Lys120, Tyr132, Arg155, and Lys161 in Fcgamma RIII and Asn120, Phe132, Ser155, and His161 in Fcgamma RI. It is, however, not clear if any of these residues contribute to the observed variation in binding affinity.


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Fig. 5.   A, sequence alignment of the membrane proximal domain of human Fc receptors. The secondary structure elements (arrow for beta -strands and squiggle for alpha -helices) are indicated under the sequence. Residues identical to the sequence of Fcgamma RIIIB are shown by periods, and gaps in sequence are shown by minus signs. 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 asterisks. B, sequence alignment of the lower hinge and CH2 regions of human IgG1, 2, 3, and 4, a mouse IgG2a and a human IgE Cepsilon 3 region. The residues contacting Fcgamma RIII are highlighted in red and blue boxes for A and B chains of the Fc, respectively.

Fc Receptor IgG Subtype Specificities-- Fcgamma receptors display IgG subtype specificities. In particular, human Fcgamma RIII binds tighter to IgG1 and IgG3 than it does to IgG2 and IgG4. Most of the Fc residues in contact with the receptor are conserved among the IgG sequences (Fig. 5B, residues boxed in blue and red), suggesting a conserved binding site for all human IgGs. These binding residues, with the exception of a Glu269 to Asp replacement, are also conserved in murine IgG2a consistent with it being a ligand for human Fcgamma receptors. The sequence differences among the IgG subclasses exist primarily at the lower hinge region. First, hIgG2 has a Val and Ala at positions 234 and 235, respectively, instead of Leu and Leu as observed in IgG1 and IgG3, and a one-residue deletion at position 237 of the corresponding IgG1. Human IgG4 has a Phe at position 234 (Fig. 5B). In addition, IgG2 and IgG4 sequences contain a three-residue deletion relative to IgG1 at the N-terminal end of the lower hinge, possibly restricting the lower hinge conformation. The length of the lower hinge has been suggested as a factor in lower receptor binding affinity of IgG2 and IgG4 (2). Among the four IgG subtypes, the length of the hinge region is longest in IgG3 and shortest in IgG2 and IgG4 (three to four residues shorter than that of IgG1). The differences in both the amino acid composition and the length of lower hinge may contribute to the observed lower receptor binding affinity of IgG2 and IgG4.

The Contribution of Carbohydrate to the Fcgamma Receptor·Fc Binding-- Both Fcgamma receptors and antibodies are glycosylated in vivo. In contrast to the Fc fragment that displays only one conserved carbohydrate attachment site located at Asn297, the receptor glycosylation sites vary both in number and in location among different Fcgamma receptors. For example, the glycosylation sites on the C-terminal domain of Fcgamma receptors are located at residues Asn162 and Asn169 on Fcgamma RIII, Asn138 and Asn145 on Fcgamma RII and asparagines 138, 145, and 149 on Fcgamma RI (Fig. 5A). 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 (4, 39). These studies demonstrated that the carbohydrate attached to Asn297 of Fc have a significant impact on the receptor binding, whereas glycosylations on the receptors appeared less critical and perhaps have more of a modulating effect on the affinity. For example, the two neutrophil antigen A alleles of Fcgamma RIIIB, NA1 and NA2, differing primarily in their carbohydrate contents, display a 2-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 Asn297 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 Asn297 of both chains of Fc toward each other into the inter-chain region, referred to as the carbohydrate core region. Asn297 is located next to the receptor binding interface. The carbohydrate moieties, however, are orientated away from the interface making no specific contacts with the receptor. The glycosylation is thus unlikely to influence the receptor·Fc interface directly. However, the unique arrangement between the oligosaccharide moieties and the polypeptide chains of Fc makes it possible for the carbohydrate to affect the conformational stability of the receptor binding epitopes (40). Specifically, the spacing and the orientation between the two CH2 domains may be influenced by the presence of sugar attachments (Fig. 1A). Because 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 CH2 domains.

A Model for Fcgamma RIII-IgG Recognition-- On a cell surface, the 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 (31). Because Fcgamma RIII 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 (Fig. 6A). This receptor·antibody recognition model reveals that the receptor fits tightly and is nearly engulfed by the bound antibody.


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Fig. 6.   Antibody-Fcgamma RIII binding and ligand induced receptor aggregation model. A, an intact antibody-Fcgamma RIII binding model. The structure of the antibody is shown in magenta and that of Fcgamma RIII is in green. The position of the second possible orientation of Fcgamma RIII, 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. B, a simple avidity model of antigen-antibody binding induced Fcgamma RIII aggregation. C, an ordered receptor aggregation model.

Although the current structure offers an insight to antibody·Fcgamma receptor recognition, the mechanism of receptor activation, namely the antigen-driven receptor clustering, remains unknown. Two receptor clustering models can be proposed based on the current structural results, a simple avidity model and an ordered receptor aggregation model (Fig. 6, B and C). The simple avidity receptor activation model assumes that the binding of oligomeric antigens by antibodies increases the avidity as well as the proximity of the receptors, which is sufficient to its activation. The ordered receptor aggregation model assumes that the binding of oligomeric antigens leads to the formation of an ordered receptor-ligand aggregation, which further stabilizes the activation complexes. Recent imaging studies on T cell and NK cell receptor activation processes suggest that the formation of the so-called immune synapse is an ordered event (41, 42). These results favor the structured aggregation model rather than the simple avidity model, although the molecular organization of Fcgamma receptors during their activation remain to be determined. Recently, an ordered receptor·ligand aggregate was observed in the crystal lattice of a natural killer cell receptor in complex with its class I major histocompatibility complex ligand (43). Such a receptor·ligand aggregate is not observed in the two forms of the current Fcgamma RIII·Fc crystals. However, a parallel receptor aggregate was observed in the crystal lattice of Fcgamma RIII in the absence of Fc (22). A superposition of the current complex structure onto this lattice receptor aggregate suggests that the clustering model be compatible with the structure of a receptor·Fc complex (Fig. 6C).

Comparison of Fcgamma Receptor with Other Ligands of Fc-- The Fc region of the IgG molecule possesses multiple recognition sites for different components of immune system, including Fcgamma 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 proteins A and G. The structures of Fc complexed to FcRn, RF, protein A, and protein G are now known (30, 33, 34, 44). The binding of Fc by Fcgamma receptors is characteristically different from all other known Fc ligands. First, the location of the Fc receptor binding site differs from those of neonatal Fc receptor, RF, and protein A. Although Fcgamma receptors bind to the lower hinge region of Fc between the CH1 and CH2 domains, FcRn, RF, protein A, and protein G bind to the joint region between the CH2 and CH3 domains of Fc. Second, Fcgamma receptors recognize Fc in an asymmetric fashion resulting in one receptor bound to both chains of Fc whereas all other ligands bind Fc in a symmetric fashion with each chain of Fc harboring an intact binding site (Fig. 7). The distinct binding site for Fcgamma receptors suggests that it is possible to bind Fcgamma receptors simultaneously with other ligands that recognize the CH2-CH3 joint region on the same Fc molecule. This raises the possibility of activation of multiple immune components by the same antigen-bound immune complex.


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Fig. 7.   Recognition of Fc by multiple ligands. Structural comparison among the complexes of (A) Fcgamma RIIIB-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 colored in cyan and shown in similar orientations. The ligands to Fc are colored in green and the beta 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.

The recognition mode of binding to the lower hinge of Fc may evolve from the unique requirement of Fcgamma receptor signaling, namely the need to have 1:1 recognition stoichiometry and to be capable of discriminating the IgG subtypes. Both CH2 and CH3 domains of Fc are very conserved among the subclasses of IgGs. Even the CH2-CH3 joint region, which is involved in binding of other Fc ligands, has near identical sequences among the IgG subclasses (Fig. 5B). The lower hinge region of IgGs, in comparison, is more variable allowing subtype-specific recognition of the receptor. The conformation of the hinge region, however, is quite flexible compared with the CH2 and CH3 domains of Fc (35). This hinge flexibility, which enables the Fab arms to adapt to the shape and form of antigens, may in fact hinder the binding of Fc receptors. Interestingly, there are two conserved cysteine residues forming two disulfide bonds at the N-terminal end of the lower hinge. The presence of these disulfides may stabilize the lower hinge conformation while allowing sufficient flexibility at the upper hinge region. Finally, the binding to the lower hinge region of both chains of Fc allows the receptor to monitor the integrity of the antibody.

Receptor-IgG Recognition and Autoimmune Diseases-- In addition to their normal cellular functions in host immunity, Fcgamma Rs, in particular Fcgamma RI and Fcgamma RIII, also mediate the inflammatory responses generated by cytotoxic autoantibodies and immune complex triggered inflammatory disorders (45, 46). They provide a critical link to autoimmune diseases, such as rheumatoid arthritis, hemolytic anemia, and thrombocytopenia. The structure of Fcgamma RIIIB 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 Fcgamma Rs. The peptides encompassing residues of the BC and FG loops of the C-terminal domain of Fcgamma receptors could also be used to develop neutralizing antibodies against the receptors. Finally, reagents that affect the glycosylation pathway may be used to affect the carbohydrate composition of Fc and thus the conformation of the receptor binding epitope.

    ACKNOWLEDGEMENTS

We thank Dr. 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 National Synchrotron Light Source X9B beam line; and Dr. S. Ginell for his assistance at the Argonne National Laboratory Structural Biology Center 19-ID beamline at the Advanced Photon Source, whose use was supported by the U. S. Department of Energy, Office of Biological and Environmental Research, under Contract No. W-31-109-ENG-38.

    FOOTNOTES

* This work was supported by the intramural research funding of NIAID, National Institutes of Health and by INSERM, Institut Curie, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Structural Biology Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-496-3230; Fax: 301-402-0284; E-mail: psun@nih.gov.

Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100350200

    ABBREVIATIONS

The abbreviations used are: Fcgamma RI and Fcepsilon RI, Fc receptors for IgG and IgE of the immunoglobulin superfamily; r.m.s., root mean square; FcRn, neonatal Fc receptor; RF, rheumatoid factors.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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