Introduction

The immune system, being the major defense complex present in vertebrates against foreign cells and pathogens, comprised an array of different specialized cells and their soluble products. The function of recognition of antigenic determinants is performed by the soluble agents, the antibodies and the diverse antigen receptors present on different cells of the immune system, all sharing features of the Multichain Immune Recognition Receptors (MIRRs) family. The MIRR activation leads to the wide range of biological responses aiming at the pathogen elimination. The structural basis of the diverse antigen recognizing receptors is derived from the random DNA editing process of the variable (V), diversity (D), and joining (J) gene segments. The result generates the diverse immune-receptor genes, whether of antibodies or B- and T-cells’ antigen receptors, taking place upon formation of a new immune cell. This yields the antigen binding site constructed of loops of hypervariable sequence which constitute the complementarity determining regions (CDRs), three from the heavy chain (VH) and three from the light chain (VL).

In the following, first kinetic studies of immunological recognition carried out by antibodies will be reviewed. This process involves stereospecific non-covalent binding of an antigenic epitope to the antibody combining site. The same type of recognition is encountered in other biochemical processes, such as enzyme and its substrate, and is accomplished through a close approach of the spatially complementary parts of the reactants with the formation of several, usually low-energy elementary interactions such as electrostatic salt bridges, hydrogen bonding, dipole–dipole interaction, charge transfer complexation and hydrophobic interaction. The recognition process, carried out by the B cells’ antigen receptors (BCRs) which are membrane anchored immunoglobulins (mIgs), is the same as that of antibodies while that of the T-cell antigen receptors (TCRs) may become more complex as it involves TCR binding to a composite structure produced by an antigen fragment, primarily peptides, bound to MHC-encoded molecules. Major efforts have been made in recent years, addressing both BCR- and TCR-ligand binding reactions and the ensuing cellular activation processes that will also be briefly addressed.

Antibody–antigen epitope interactions

Polyclonal antibodies–hapten interaction kinetics

The pioneering kinetic studies of hapten binding to polyclonal hapten-specific antibodies by chemical relaxation have been carried out by Froese and Eigen. This method causes perturbation of the antibody–hapten equilibrium by a rather fast rise in temperature, enabled resolution of the relatively fast relaxation to the new state as monitored by the absorption or fluorescence changes of the reactants and/or products. Only a single relaxation step has been observed suggesting the operation of a single-step binding mechanism:

$${\text{Ab}} + {\text{H}}\mathop{\rightleftarrows}\limits_{{\text{k}}_{21}}^{{\text{k}}_{12}}{\text{Ab}} \cdot {\text{H,}}$$
(1)

where Ab, H, and Ab·H are the antibody, the hapten, and their complex, respectively, while the specific rates of association are k12 and dissociation, k21, respectively (Froese et al. 1962; Day et al. 1963).

Kinetic mapping of a monoclonal antibody binding site

Availability of the very first monoclonal antibodies, secreted by induced myeloma cells enabled the first study of a homogeneous antibody and a more rigorous kinetic analysis of the process. The myeloma-secreted monoclonal, nitroaromatic haptens’ specific IgA-MOPC315 antibody has therefore been the first to be investigated and by chemical relaxation. Again, only a single relaxation process has been observed for the reaction suggesting operation of a single-step equilibrium (Pecht et al. 1972a, b). This led to an extensive kinetic study of a large number of systematically varied hapten structure interacting with this binding site. While the rates of the binding step approached the diffusion controlled limit and varied within less than one order of magnitude, the dissociation rates differed by more than two orders, reflecting specific elementary interactions between the contact residues of the site and different parts of the hapten. Using these detailed kinetic parameters enabled the so-called ‘kinetic mapping’ of the site reflecting specific interactions between the hapten and its binding site (Pecht et al. 1972a, b; Haselkorn et al. 1974).

It is, however, significant that later, computer-assisted analysis of the chemical relaxation spectrum of hapten binding kinetics by this monoclonal antibody (mAb) MOPC315 was found to exhibit a second, additional relaxation phase of rather small amplitude that could not be resolved by the earlier available data analysis method yet did not markedly affect earlier conclusions. Thus, the original assumption that binding site–epitope interactions could be represented by the single-step equilibrium [Eq. (1)] with a diffusion-controlled rate of binding and having the dissociation rate constants be the main reflection of the binding affinity had been limited.

Conformational transition(s) accompany antibody–hapten interactions

Indeed, already in the mid 1970s studies of second available myeloma secreted mAb, MOPC460 was found to display two distinct relaxation times upon reaction with its nitroaromatic ligand. The first in the range of 0.25–1.0 ms and the slower, in the range 10–18 ms (Lancet and Pecht 1976). Detailed analysis of the well-resolved relaxation spectrum of MOPC460 interactions with its hapten, its amplitudes and their dependence of reactants’ concentrations, suggested the mechanisms summarized by Fig. 1. Namely, this antibody site exists in two conformational states T and R with an equilibrium between them in the free state. Upon hapten binding, the equilibrium is shifted to a higher affinity binding state.

Fig. 1
figure 1

The above formulas present the possible reaction patterns of antibody–epitope interactions, involving conformational transitions of the Ig binding site. H represents the epitope; R0 and R1 free and bound antibody with higher affinity; T0 and T1 of the respective lower one

Full size image

The results of the kinetic studies of MOPC460–hapten interactions have later on received interesting complementation from investigation of this system by nuclear magnetic resonance spectroscopy (Morris et al. 1980). In this study, interactions with three different dinitrophenyl (DNP) derivatives have been pursued, monitoring perturbation of the mAb’s aromatic amino acid residues by the bound haptens. The NMR results have yielded further support to the above model proposed by Lancet and Pecht for the mAb–hapten interactions. Chemical shifts of residues assigned to the binding sites suggest that the geometry of those interacting with the haptens is different in the T and R conformers of the complex. This was further supported by results of measuring the hapten’s resonances indicating distinct hapten-binding environments within the two conformers.

Conformational diversity is observed in more Ig classes and with different specificities

As increasing number of mAbs of different specificities and classes became available, further kinetic studies made it quite clear that epitope binding-induced conformational transitions are a general characteristic of antibodies’ binding sites. Following the first kinetic study describing the hapten binding-induced conformational transition in MOPC460 (Lancet and Pecht 1976), the systematic kinetic study was extended to a number of other monoclonal antibodies of diverse specificities and belonging to classes other than IgA. A common mechanism was consistently found to govern these reactions. In the following are a few illustrative cases.

A chemical relaxation T-jump study of saccharide ligands binding kinetics to a mAb raised to Pneumococcal type III oligosaccharides has been published in Maeda et al. (1977). Interactions between di-tetra and hexa-saccharides and the IgG class mAb 45-394 have been monitored by the intrinsic protein fluorescence. Significantly, though only a single relaxation time could be observed for interactions of any of the studied oligosaccharides, detailed analysis of the relaxation times obtained for all three could be rationalized only by assuming the existence of a slow conformational equilibrium of the initially produced labile mAb–hapten pre-complex.

The availability of effective protocols for the production of monoclonal antibodies by hybridomas enabled more kinetic studies of the interactions between raised mAbs of known specificity with their ligands. Early insights that have been based primarily on work employing chemical relaxation measurements were later complemented also using stopped-flow measurements. The time-resolved analysis of hapten-induced conformational transitions, though suffering at that stage from limited knowledge of their structural and functional significance, have provided more evidence for the existence of multiple conformational states of antibody binding sites.

Kinetics of interaction between the murine IgM-class mAb MOPC104E specific for α(1–3)-dextran with a series of α-D-glucopyranosyl-(1-3) oligomers of different sizes and structures with this antibody were investigated. The observed chemical relaxation spectrum was found to exhibit two well-resolved relaxation times for interactions with all examined ligands (Schepers et al. 1978). Analysis of both the relaxation times’ and amplitudes’ concentration dependence of interaction between MOPC104E with the tetra-saccharide was shown to fit the same general mechanism formulated earlier for MOPC 460 (Fig. 1).

Another system studied extensively by the chemical relaxation method was the interaction of three different galactan-specific IgA class mAbs XRPC24, J539, and TEPC601 with their oligo-galactose haptens. Reactions were again monitored by changes in the intrinsic fluorescence of the proteins. The chemical relaxation spectra of all three mAbs reacting with (Gal)3 were found to consist of two relaxation processes (Vuk-Pavlovic et al. 1978; Zidovetzki et al. 1980). A detailed analysis of the concentration dependence of the relaxation times and amplitudes has also shown that this system behaves according to the same general mechanism above.

Significantly, kinetic results of all the above different systems were obtained using both, the intact immunoglobulins as well as their Fab fragments. All systems were found to exhibit identical kinetic behavior, clearly indicating that the mAb-ligand binding reaction mechanism, including the conformational transitions, is not affected by the Ig-Fc domain.

Binding sites’ conformational flexibility may also be observed in hybrid antibodies

To try examining further the generality of the mechanism observed for practically all mAbs investigated by that time point, challenging studies have been undertaken of the kinetics of hapten interactions with a series of heterologous recombinants of heavy and light chains prepared from the above described family of galactan-specific antibodies (X24, J539, and T601) (Zidovetzki et al. 1980). This group of recombinant mAbs had earlier been shown to maintain an affinity for the β-D-(1->6)-oligogalactose haptens comparable to that of their parent molecules (Manjula et al. 1976). The kinetics of the hybrid antibody–hapten interactions were also found to exhibit two relaxation times. The reaction rates and amplitudes data for the hybrids were found to fit the same general mechanism as that followed by their parent proteins, namely, the hybrid mAbs were shown to exist in two conformational states with their equilibria shifted to the higher affinity state on hapten binding. Furthermore, some of the specific rates and the thermodynamic parameters of these different steps were found to have values very close to those of their parent molecules.

The kinetic and thermodynamic parameters obtained for this group of related antibody molecules were then examined in the context of specific differences in their binding site amino acid sequences. Particularly interesting has been the comparison between each individual hybrid and its parent mAbs: a significant correlation with the parent light chain donor was found only for the rates of conformational transition of the hapten-bound state (k1 and k-1) of the hybrids. This observation is instructive as the light chains of T601 and X24 have identical sequences except for a single alanine exchanged for a serine at position 100. mAb J539 differs from the former at five positions, and from the latter at six positions (Rao et al. 1979; Rudikoff et al. 1980). The VH regions of X24 and T601 differ at six positions, three of which are clustered in the J segment. VH of J539 differs from that of the two previous ones in a larger number of positions, both in the D and J segments, as well as in the rest of this domain (Rao et al. 1979). Examinations of the positions have shown that the substitutions in the chains constituting these hybrids occupy in the three-dimensional structure of their domains are rather informative: they show that several of the more important exchanges (e.g., Ser to Ala at L-100) are in the VL–VH contact areas (Feldman, personal communication 1980). This is in line with the J segment having a decisive role in the light chain folding, and most probably, also in the heavy light chain association. Thus, the recombination events joining VL with J1 and VH with D and JH, apart from affecting directly the chemical nature and morphology of the antigen combining site, because of their presence at the VL-VH interface, they also modulate it.

A further interesting result of these studies is that the rates of the structural transitions of the hapten-bound mAb exhibit the widest variation, spanning more than four orders of magnitude (cf. Table VII, Pecht 1982). A correlation that apparently reflects differences in site–hapten interactions.

The mAb–hapten association rate constants of these group are similar to those observed for other saccharide-binding proteins (Clegg et al. 1977; Pecht 1976) and are two orders of magnitude slower than the association rate constants observed for small rigid haptens such as nitroaromatic molecules binding to their specific antibodies (Haselkorn et al. 1974; Pecht and Lancet 1977). Even slower bimolecular rate constants were determined for the binding of saccharide to lectins (Clegg et al. 1977; Loontiens et al. 1977). As suggested earlier (Pecht and Lancet 1977; Vuk-Pavlovic et al. 1978), these slow rates of saccharide (and peptide) binding to proteins may be a result of their flexibility and/or the requirement to disrupt and form several hydrogen bonds on transition from the encounter complex to the final complex formation step.

Kinetic analysis of antibodies’ affinity maturation

The above studies were motivated by the basic interest in the mechanism of mAb-antigen reaction being a general model for protein–ligand interaction kinetics as well as the antigen recognition process. An additional, related fundamental question also led to a detailed study of this reaction’s kinetics. Foote and Milstein (1991), in their investigation of humoral immune maturation in mice, have examined the kinetics of the hapten 2-phenyl-5-oxazolone binding to a family of 40 different mAbs raised against it. Specifically, the analysis of mAbs produced at different stages of immune response to this given hapten was pursued to determine what parameters, thermodynamic, kinetic or both control the maturation of antibody affinity? This study employed the stopped-flow method for kinetic measurements of hapten-binding of the hybridoma-secreted mAbs, at different stages of immune response maturation. A shift towards a relatively high hapten association rate constant was observed and suggested to underlie the increased affinity of the secondary and tertiary response repertoires. However, only a limited analysis of the kinetics results had been carried out. Assuming the interactions to be single-step equilibria, the dissociation rates have not been determined directly but rather calculated from the equilibrium binding constants. Furthermore, the use of a relatively rigid hapten might have led to the former interpretation, assigning affinity increase to the observed increase in the association rates.

A later study of three mAbs (out of the 40 examined earlier) that were found to exhibit additional reaction steps has led the authors to address another fundamental question, namely whether a single binding site sequence results, upon its folding, in a single three-dimensional structure? (Foote and Milstein 1994). This question arose first from results of increasing reports of kinetic studies, also summarized above, providing evidence for conformational transitions between distinct isomers of antibody sites. In addition, crystallographic evidence has also slowly yielded evidence for the existence of structural differences in mAbs’ binding sites between the free and occupied states. Further kinetic analysis of the 2-phenyl-5-oxazolone hapten binding process to these three mAbs have shown it to exhibit bi-or tri-phases of binding as monitored by stopped-flow. This has indeed yielded further independent support for the existence of conformational transitions among distinct structures of the antigen binding site. Implications of these findings led to several predictions such as having evolution of V genes likely to exhibit such isomerism to increase its diversity. In addition, site isomerism could lead to increase in binding affinity and may mediate recognition of dissimilar antigenic epitopes, i.e., yielding bi-specificity of a one given mAb.

A different trend in mAb site properties evolution was discovered in a study of an antibody combining site involved in catalytic activity. It examined the sequence and three-dimensional structures’ differences between sites of hapten-specific germline mAb in the free and complexed states with those of an affinity-matured mAb of the same specificity (Wedemayer et al. 1997). Binding site of the germline mAb resolved significant changes in structure occurring in the site configuration upon binding the hapten. In contrast, the mature site having more than four orders of magnitude higher affinity had an essentially rigid lock-and–key fit of the site and its hapten. These results support the notion that binding sites of a primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt multiple conformations while further somatic mutations during evolution of the response to the hapten, yield a combining site with improved complementarity higher affinity and apparently rigid site–ligand interactions.

Conformational diversity may yield antibody multi-specificity

A most innovative study further establishing the significance of conformational plasticity of antibody binding sites has been that of James et al. (2003). This study combined several approaches, including kinetic and structural to an in-depth investigation of mAb-antigen binding characteristics. An IgE class mAb originally raised against the 2,4 dinitrophenyl hapten was shown to have a binding site with a pre-existing equilibrium between several distinct conformations. Thereby, it was shown to bind distinct, structurally unrelated antigenic epitopes. As in other cases, the epitope-induced conformational transition that led to a higher affinity complex formation. Furthermore, as suggested before, the possible presence of multiple site structures can increase the effective size of the antibody recognition repertoire.

This notion that an antibody binding site can adopt different conformations, thereby markedly increasing the capacity of different specificities as well as attaining higher affinities is now widely appreciated.

Coupling antigen recognition to functional response

Is conformational flexibility limited to the Ig epitope binding site?

In parallel to the kinetic studies, the continuously growing database of 3D structures of free and ligand-bound antibodies yielded further insights into the nature of binding site interactions with its antigenic epitope and changes in it. Changes in the sites were found to range from limited fluctuations of the epitope-contacting residues to those involving gross domain movements (Wilson and Stanfield 1994; Sundberg and Mariuzza 2003). In the latter case, flexibility in the junction linking the V and C super domains as defined by the Fab elbow angle was found in equal frequency for both free and ligand-bound structures. Recently, a rather thorough systematic comparison of the 3-D structures of free and bound antibodies available in the PDB database has been carried out (Sela-Culang et al. 2012, 2013). 141 structures of 49 mAbs that were determined in both, their free and bound states were compared. Results of this comparison have shown that within the binding sites, CDR-H3 exhibits most pronounced induced changes upon antigenic epitope binding, though observed in only a third of the examined structures. Significantly, antigen binding was found to be associated with changes in the relative orientation of the H and L chains in both variable and constant domains. Largest change was observed in the elbow angle between the latter domains, and the most consistent and substantial changes were found to occur in one H chain constant domain loop. A loop implicated in the H and L chain interactions which might therefore be involved in coupling epitope binding to Ig effector functions. These results and a considerable number of recent reports brought back to the forefront discussions of the allosteric mechanism for the induction of effector functions of antibodies by antigen binding, originally proposed by Huber et al. (1976). Indeed, consistent spectroscopic evidence for antigen binding-induced structural changes had accumulated from early studies employing measurements of antibodies Circularly Polarized Luminescence (CPL) (Jaton et al. 1975; Schlessinger et al. 1975; Givol et al. 1977). This method is the fluorescence analog of circular dichroism as it monitors the circular dichroism of a chromophore’s excited state. In several studies, the CPL of different Abs intrinsic, predominantly tryptophans’ circular polarized fluorescence, in the free and antigen-bound states, exhibited marked changes assigned to structural changes caused upon binding. Detailed examination of these spectroscopic results obtained for intact, Fab and reduced Ig hinge disulfide bridges suggested that the antigen-induced structural changes may be transmitted all the way to the Fc domains, which upon antigen-induced clustering provide the oligomeric (hexamer) structure that is required for complement activation (Diebolder et al. 2014; Lee et al. 2017). Interestingly, another early study that combined CPL and complement activation measurements yielded evidence for such structural changes being correlated with the activation of complement (Pecht et al. 1977).

Thus, one still encounters a major challenge to acceptance of an allosteric mechanism being the requirement that structural changes be communicated to the Fc domains via the flexible Ig hinge region. Hence, the mechanism whereby those immunoglobulin effector functions involving the Fc domains are initiated upon antibody–antigen complex formation was based on the observation that the latter process causes antibodies aggregation. This led to the still prevailing model for the initiation of activation process being the formation of immunoglobulins clusters, whether in solution or on cell membranes (“Immunoglobulins as Fc receptors antigen recognition and activation elements” section below).

Immunoglobulin as a component of the B-cell antigen receptor

One line of research aiming at resolving the mechanism of coupling Ag recognition with activation of effector functions focused on B lymphocytes. In these cells, the antigen-recognizing element is their membrane-anchored immunoglobulin (mIg). Recent extensive studies of Reth and his associates brought evidence for a novel, rather elaborate and challenging model for BCR activation, markedly different from the above involving Ig aggregation: the new model is based on results obtained from a range of different experimental approaches. It suggests that on resting B cells’ membranes the BCRs exist as inactive, (self-inhibited) oligomers. Antigen binding to the BCRs is opening the pre-organized oligomers and thereby initiate the signaling cascade coupling it to cell’s response (Yang and Reth 2010a, b; Fiala et al. 2011; Fiala et al. 2013; Klaesener et al. 2014; Volkmann et al. 2016). Based on these and other results Yang and Reth provide compelling arguments criticizing the so-called cross-linking activation model requiring Ig aggregation. Their model excludes the requirement of initial receptor crosslinking as the activating signal. Rather surprising, their studies have also presented results suggesting that even monovalent antigens are capable of perturbing and opening inactive, (self-inhibited), oligomers. In other words, monovalent agents are incapable of cross-linking the BCRs yet have also been found to cause activation. Still, significant differences were observed in activating capacities of the polyvalent and different monovalent agents, primarily differences in response amplitudes. Interesting are the differences in the nature of interactions between the monovalent agents and their BCR: only those ligands that interact with mIg binding site of the BCR, such as hapten derivatives or Fab fragments of anti-idiotype antibodies that bind to antigen binding site residues of the mIg were found to be effective.

Independent evidence for the observed capacity of monovalent ligands to activate upon binding to the BCR has in fact been reported earlier by the Ploegh lab (Avalos et al. 2014). They investigated the ligand valence requirements for BCR activation using ovalbumin epitopes. A very detailed examination of different structures, length and valence of synthetic peptides derived from ovalbumin has provided evidence for the activating capacity of monovalent ligands. Among different methods employed for studying the activation process, binding measurements of different ligands to the mIg using surface plasmon resonance (SPR) were carried out (Avalos et al. 2014). Unfortunately, the inherent drawbacks of the latter method have not been taken in account in analyzing the data: first, results of SPR measurements do not necessarily reflect the actual elementary steps of the examined process, and in addition, the method does not provide the required time resolution. An illustration of the complexity and caution required in investigating kinetics of ligand binding to immune receptors have been provided by a study where the T-cell receptor interaction kinetics with its specific ligand were investigated while comparing results of using both SPR and stopped-flow methods (Gakamsky et al. 2007). Conformational equilibria of antigen binding sites, preceding the actual site–ligand complex formation step were found to be a common mechanistic feature also in the present case.

As elaborated above, evidence is still missing for an Ag binding-induced conformational transition transmitted longitudinally to the Fc domains. Hence, the observations that monovalent antigens are capable of opening pre-existing auto-inhibited BCR oligomers and inducing cell activation needs an alternative mechanism. An attempt to provide a rational has been brought up in a study of functional differences in the hinge region and constant domains flexibility of IgD- and IgM-containing BCRs (Ueberhalt et al. 2015): it suggested that mIgM-containing BCRs may undergo low-affinity binding with an additional epitope present on an apparent monovalent antigen, thus functionally acting as a polyvalent one. However, examination of the data does not support this hypothesis as even monovalent haptens do exhibit activating capacity (Volkmann et al. 2016). Hence, one may consider an alternative rational. Namely, that the auto-inhibited oligomeric BCRs are pre-organized in such a manner that their antigen binding sites are packed rather densely. Then the conformational transition induced in the binding site upon binding even its specific hapten, and probably more so, binding an intact antigen or a CDR-specific anti-idiotype Fab, will cause perturbation of the latter, auto-inhibited oligomeric structure, leading to its rearrangement and cell activation. This possible model may suggest that the interactions between mIg site and its antigen ligand might not be limited to the recognition process but could be also involved in initiating the trans-membrane signaling cascade itself.

Immunoglobulins as Fc receptors antigen recognition and activation elements

For Fc receptors, antibodies fulfill the role of antigen recognition. The nature of the activation signal produced by Fc receptor bound antibodies has been studied extensively on mast cells as a model system where the type 1 Fcϵ receptor (FcϵRI) bound IgE initiates cell secretory responses upon reaction with antigens. Thus, the very fact that the FcϵRI undergoes aggregation by antigen served as the main support for the receptor clustering model for cell activation (Wilson et al. 2011).

One early effort to quantitatively examine the requirements for activation via the type 1 FcϵR employed several mAbs raised against its alfa subunit. Fab fragments of these IgG class mAbs were shown to bind each in a stoichiometric manner to the receptor and cluster it into dimers (Ortega et al. 1988). Quantitative comparison of cells’ secretory response with the number of dimers produced by each of the different mAbs has shown that while dimer formation is required and sufficient for stimulation, the cells secretory response did not correlate with the number of produced dimers. Namely response to dimers differed markedly depending on the dimer producing mAb. Two possible rationales were examined for these observations: first was differences in the dimers’ life times. Kinetics of dimer formation and dissociation excluded this rational (Schweizer-Stenner et al. 1994). Hence the alternative suggested to rationalize the different observed responses was that different structural constraints are imposed on the FcϵRIs by the different mAbs yielding dimers with distinct orientation with respect to each other. Considering the rather asymmetric structure of all MIRRs in general, and the FcϵRI in particular, the relative spatial relations between closely clustered receptors would be a key determinant for producing the activation signal. This idea is in line with the above detailed model proposed for the initiation of BCR activation requiring their close and optimal orientational alignment for producing the signaling trigger.

The results obtained using receptor-specific mAbs led to an interesting experimental protocol aiming to quantitatively investigate the FcϵRI proximity, cluster size, and mobility requirements for mast cell stimulation. To this end, mast cells were reacted with glass surfaces carrying well-controlled different densities of covalently bound antigen and IgE, and the cell’s secretory response to these stimuli was evaluated (Tamir et al. 1996; Schweitzer-Stenner et al. 1997). Results have established that sustained FcϵRI secretory response requires solely that the average density of immobilized FcϵRI exceed a distinct threshold value for a period of time that allows stimulus–secretion coupling to take place. In addition to providing information regarding receptor density requirements for stimulation, it has also established the limited need for receptor motion. Importantly, this protocol also provides a model system for examining cell–cell interactions leading to stimulation, e.g., antigen presentation to the BCR by another cell, yielding activation.

Conclusions

Understanding how effector functions of immunoglobulins are activated upon antigen binding is still a topic of considerable interest whether they serve as antibodies, the soluble arm of the adaptive immune response or as cell bound, to specific Fc receptors or membrane anchored part of the B-cell receptors. Current knowledge is that the antigen combining site of immunoglobulins can exhibit conformational diversity, providing multiple specificity and increased affinity. This conformational variability is concentrated primarily in the CDRs. Antigen binding-induced conformational changes were, however, also identified beyond the variable domains yet the question whether they are conveyed all the way to the Fc domains of immunoglobulins is still open. Thus, while earlier models of effector function activations were all based on Ig clustering, currently allosteric models are revived. Notably, the earlier model of B-cell activation by antigen clustering of its mIg has recently been seriously challenged: a large body of experimental evidence supports the model where BCRs exist in the cell membranes in a pre-clustered, auto-inhibited state, that undergoes configurational rearrangement upon antigen binding and this latter process initiates the cellular activation cascade. By analogy, proximity and orientational constraints may also constitute the trigger of signaling by Fc receptors.