FG Loop Research Articles

Involvement of the TCR Cbeta FG loop in thymic selection and T cell function.

The asymmetric disposition of T cell receptor (TCR) Cβ and Cα ectodomains creates a cavity with a side-wall formed by the rigid Cβ FG loop. To investigate the significance of this conserved structure, we generated loop deletion (βΔFG) and βwt transgenic (tg) mice using the TCR β subunit of the N15 CTL. N15βwt and N15βΔFG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4−CD8− double-negative (DN) compartment. N15βΔFG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2−/− mice, showing that pre-TCR function remains. N15βΔFG animals possess ∼twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Vα repertoire observed in N15βΔFG mice may confound the deletion's effect, we crossed N15αβ TCR tg RAG-2−/− with N15βΔFG tg RAG-2−/− H-2b mice to generate N15αβ RAG-2−/− and N15αβ.βΔFG RAG-2−/− littermates. N15αβ.βΔFG RAG-2−/− mice show an 8–10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15αβ, N15αβ.βΔFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cβ FG loop facilitates negative selection of thymocytes and activation of T cells.

Crystal structure of murine sCEACAM1a[1,4]: a coronavirus receptor in the CEA family.

CEACAM1 is a member of the carcinoembryonic antigen (CEA) family. Isoforms of murine CEACAM1 serve as receptors for mouse hepatitis virus (MHV), a murine coronavirus. Here we report the crystal structure of soluble murine sCEACAM1a[1,4], which is composed of two Ig-like domains and has MHV neutralizing activity. Its N-terminal domain has a uniquely folded CC′ loop that encompasses key virus-binding residues. This is the first atomic structure of any member of the CEA family, and provides a prototypic architecture for functional exploration of CEA family members. We discuss the structural basis of virus receptor activities of murine CEACAM1 proteins, binding of Neisseria to human CEACAM1, and other homophilic and heterophilic interactions of CEA family members.

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between β-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses.

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.

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

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 (FcgammaRIIIB) in complex with an Fc fragment of human IgG1 determined from orthorhombic and hexagonal crystal forms at 3.0- and 3.5-A 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 FcgammaRIII, including the BC, C'E, FG loops, and the C' beta-strand, bind asymmetrically to the lower hinge region, residues Leu(234)-Pro(238), 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 FcgammaRIII.Fc and FcepsilonRI.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 Asn(297) on Fc may stabilize the conformation of the receptor-binding epitope on Fc. An antibody-FcgammaRIII model suggests two possible ligand-induced receptor aggregations.

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-α-1,2-dihexanoylphosphatidylcholine/l-α-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.

Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells.

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.

Pregnancy-Specific Glycoprotein (PSG) in Baboon (Papio hamadryas): Family Size, Domain Structure, and Prediction of a Functional Region in Primate PSGs1

Pregnancy-specific glycoprotein (PSG) constitutes a major component of serum of pregnant women and appears to be essential for a successful pregnancy. Its function is, however, still unknown. Because of the evolutionary divergence between human and rodent PSG, functional studies may require a primate animal model. We have characterized PSG transcripts in a baboon placenta cDNA library and analyzed baboon genomic DNA. The main PSG isoform had the domain structure N-A1-B2-C similar to the human type IIa isoform. The type I isoform (N-A1-A2-B2-C) was also expressed. Fifteen similar PSG genes were identified of which at least nine were simultaneously expressed in third trimester baboon placenta. Thus, the baboon PSG family was as complex as that of humans. Recombinant baboon PSG (isoform IIa) had a molecular weight of 38 kDa and reacted with antibodies against human PSG. Comparative analysis of 43 N-domain amino acid sequences of PSG from four species and nine primate carcinoembryonic antigen subgroup N domain sequences identified a number of residues in the GFCC'C" ss-sheet and FG loop that are probable candidates for PSG binding to its putative ligand.

Dimeric Structure of the Coxsackievirus and Adenovirus Receptor D1 Domain at 1.7 Å Resolution

Background: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. Results: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 Å resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C″D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 μM. Conclusions: The CAR D1 domain forms homodimers in the crystal using the same GFCC′C″ surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.

Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods

Icosahedral virus-like particles (VLPs) of RNA phage Qβ are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between β-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs. In order to reduce the stability of Qβ VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like Qβ VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4–30 times the diameter of icosahedral Qβ VLPs.

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

Functional Significance of Hierarchical Tiers in Carbonmonoxy Myoglobin: Conformational Substates and Transitions Studied by Conformational Flooding Simulations

The functional importance of large-scale motions and transitions of carbonmonoxy myoglobin (MbCO) conformational substates (CSs) has been studied by molecular dynamics (MD) and conformational flooding (CF) simulations. A flooding potential was constructed from an 800 ps MD trajectory of solvated MbCO to accelerate slower protein motions beyond the time scale of contemporary simulations. Two conformational transitions (tier-1 substates) resulting from seven principal molecular motions were assigned to the spectroscopic A0 state (tier-0 substate) of MbCO, where His64 is solvated and not within the hydrophobic pocket binding site. The first computed conformational transition involves a distal pocket gate defined by the C and D helices and the interconnecting CD loop (residues 40−55). The gate-like motion is interpreted to regulate ligand access from the distal side of the heme. Simultaneously, a proximal pocket lever involving the F helix and surrounding EF and FG loops (residues 82−105) is found to shuttle the heme deep into the protein matrix (heme rmsd of 3.9 Å) as the distal pocket gate opened. The lever's effect on the heme motion is assumed to attract ligands into the heme pocket. The second major transition involves the compression and expansion of the cavity formed by the EF loop (residues 77−84) and the GH loop and H helix (residues 122−138). The motion is interpreted to modulate the hydrophobic pocket volume and regulate the ligand access from the proximal side of the heme. A third computed conformational transition was found to be a combination of the previous motions. For the first time, CF was applied in a series of room temperature simulations to accelerate molecular motions of the MbCO native fold and define the lower tier hierarchy of substate structure. The computed CSs and associated transitions coincide with previously suggested putative ligand escape pathways, and support a hierarchical description of protein dynamics and structure. A unified model that utilizes both mechanisms of distal His64 modulation (tier-0) and protein equilibrium fluctuations (tier-1) is presented to explain ligand diffusion in the MbCO dissociation reaction.

The Three-Dimensional Structure of Bacteriophage PP7 from Pseudomonas aeruginosa at 3.7-Å Resolution

The three-dimensional structure of phage PP7 from Pseudomonas aeruginosa has been determined to 3.7-Å resolution. A comparison with distantly related small RNA phages showed that the biggest differences were found in the FG loops, forming the contacts around the fivefold and threefold axes. In contrast to the situation in other phages, the FG loops of phage PP7 are very similar in all three subunits. This supports the hypothesis that no switches are needed for the assembly control in these viruses. Some of the most conserved residues lie within the region involved in RNA binding in the related phages MS2 and seem to have the same function.

IDENTIFICATION OF REGIONS WITHIN THE THIRD FnIII-LIKE DOMAIN OF THE IL-5Rα INVOLVED IN IL-5 INTERACTION

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Rα). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Rα (hIL-5Rα) and mouse IL-5Rα (mIL-5Rα) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Rα but not the mIL-5Rα. By using chimeras of the mIL-5Rα and hIL-5Rα we demonstrate that residues within the first and third FnIII-like domains of mIL-5Rα are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Rα to mIL-5Rα within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Rα are involved in binding, in addition to the regions in domains one and two of the IL-5Rα that were identified in an earlier study.

Ordered membrane insertion of an archaeal opsin in vivo.

The prevailing model of polytopic membrane protein insertion is based largely on the in vitro analysis of polypeptide chains trapped during insertion by arresting translation. To test this model under conditions of active translation in vivo, we have used a kinetic assay to determine the order and timing with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon Halobacterium salinarum. BO is the apoprotein of bacteriorhodopsin, a structurally well characterized protein containing seven transmembrane alpha-helices (A-G) with an N-out, C-in topology. H. salinarum strains were constructed that express mutant BO containing a C-terminal His-tag and a single cysteine in one of the four extracellular domains of the protein. Cysteine translocation during BO translation was monitored by pulse-chase radiolabeling and rapid derivatization with a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The results show that the N-terminal domain, the BC loop, and the FG loop are translocated in order from the N terminus to the C terminus. Translocation of the DE loop could not be examined because cysteine mutants in this region did not yield a gel shift. The translocation order was confirmed by applying the assay to mutant proteins containing two cysteines in separate extracellular domains. Comparison of the translocation results with in vivo measurements of BO elongation indicated that the N-terminal domain and the BC loop are translocated cotranslationally, whereas the FG loop is translocated posttranslationally. Together, these results support a sequential, cotranslational model of archaeal polytopic membrane protein insertion in vivo.

Review: Rhinoviruses and Their ICAM Receptors

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen or macrophage-1 antigen. However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses.

Impaired NK1.1 T cell development in mice transgenic for a T cell receptor beta chain lacking the large, solvent-exposed cbeta FG loop.

A striking feature of the T cell receptor (TCR) beta chain structure is the large FG loop that protrudes freely into the solvent on the external face of the Cbeta domain. We have already shown that a transgene-encoded Vbeta8.2(+) TCR beta chain lacking the complete Cbeta FG loop supports normal development and function of conventional alpha/beta T cells. Thus, the FG loop is not absolutely necessary for TCR signaling. However, further analysis has revealed that a small population of alpha/beta T cells coexpressing NK1.1 are severely depleted in these transgenic mice. The few remaining NK1.1 T cells have a normal phenotype but express very low levels of TCR. We find that the TCR Vbeta8.2(+) chain lacking the Cbeta FG loop cannot pair efficiently with the invariant Valpha14-Jalpha281 TCR alpha chain commonly expressed by this T cell family. Consequently, fewer NK1.1 T cells develop in these mice. Our results suggest that expression of the Valpha14(+) TCR alpha chain is particularly sensitive to TCR-beta conformation. Development of NK1.1 T cells appears to need a TCR-beta conformation dependent on the presence of the Cbeta loop that is not necessarily required for assembly and function of TCRs on most alpha/beta T cells.

The structure of the two amino-terminal domains of human intercellular adhesion molecule-1 suggests how it functions as a rhinovirus receptor

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-l). However, ICAM-1 is also utilized as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 Å resolution and fitted into a cryo-electron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE and FG loops of the amino-terminal immunoglobulin-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1 which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the I-(insertion) domain on the α chain of LFA-1 and the carboxy group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

T cell receptor beta chain lacking the large solvent-exposed Cbeta FG loop supports normal alpha/beta T cell development and function in transgenic mice.

The striking and unique structural feature of the T cell receptor (TCR) beta chain is the bulky solvent-exposed FG loop on the Cbeta domain, the size of almost half an immunoglobulin domain. The location and size of this loop suggested immediately that it could be a crucial structural link between the invariant CD3 subunits and antigen-recognizing alpha/beta chains during TCR signaling. However, functional analysis does not support the above notion, since transgene coding for TCR beta chain lacking the complete FG loop supports normal alpha/beta T cell development and function.

Identification of Residues in the First Domain of Human Fcα Receptor Essential for Interaction with IgA

AbstractThe FcR family contains multiple receptors for Igs, of which the most distantly related (∼20%) is the IgA receptor (human FcαR), being more homologous (∼35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the FcαR demonstrated that, like several IgG receptors, FcαR is a low affinity receptor for Ab (Ka ∼ 106 M−1). Rapid dissociation of the rsFcαR:IgA complex (t1/2 ∼ 25 s) suggests that monomer IgA would bind transiently to cellular FcαRs, while IgA immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the IgA-binding activity of FcαR, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (FcγRII, FcγRIII, and FcεRI), in which the ligand binding site is in domain 2 rather than domain 1, but like FcαR, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the FcαR ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.