DOI: 10.5061/dryad.6q573n66m
title: Functional evolution of antibody Fc domains: SAXS plots and models

description: Introduction: The Fc regions of antibodies contain binding sites
 for receptors that facilitate effector functions after the Fab regions
 engage with antigens. The IgG antibody features an extended
 "hinge" region that provides flexibility between the Fab and Fc
 domains. In contrast, both the more primitive IgM and the more
 evolutionarily recent IgE replace this hinge with an additional pair of
 domains within the homo-dimeric, six-domain Fc structure. This structural
 adaptation allows for increased flexibility within the Fc region, which
 nature has leveraged to regulate antibody effector functions. In the case
 of pentameric or hexameric IgM, the Fc regions adopt a planar conformation
 in solution until antigen binding induces a conformational change that
 reveals complement binding sites. Conversely, IgE-Fc predominantly exists
 in a sharply bent conformation in solution, with receptor binding
 modulated by the extent of this bend and featuring allosteric interactions
 between receptor binding sites. Methods: To investigate the
 evolutionary trajectory of Fc conformational diversity from IgM to IgE
 through the intermediary avian IgY, we employed small-angle X-ray
 scattering to assess the solution conformations of their Fc regions. Our
 study focused on four extant proteins: human IgM-Fc homo-dimer, chicken
 IgY-Fc, platypus IgE-Fc, and human IgE-Fc. These proteins represent
 evolutionary milestones: originating in jawed fish (425 million years
 ago), tetrapods (310 million years ago), monotremes (166 million years
 ago), and hominids (2.5 million years ago), respectively. Results and
 Discussion: We analyzed the scattering curves by evaluating
 contributions from a set of variously bent models identified through a
 non-negative linear least-squares algorithm. Our findings reveal a
 progressive increase in the proportion of acutely bent structures among
 the proteins: IgM-Fc &lt; IgY-Fc &lt; platypus IgE-Fc &lt;
 human IgE-Fc, aligning with their evolutionary sequence. Human IgM-Fc
 homo-dimer exhibits no acutely bent structures; however, a notable portion
 of the protein is sufficiently bent to unveil the C1q binding site while
 predominantly maintaining a fully extended conformation. In contrast,
 human IgE-Fc is primarily acutely bent, consistent with previous studies.
 The IgY-Fc, presented here in the first complete structural analysis of
 its Fc region, displays a range of conformational states from acutely bent
 to fully extended, underscoring its role as an evolutionary intermediary
 between IgM and IgE.
description: <strong>Introduction</strong>
 Throughout the evolution of antibodies from the emergence of IgM
 in jawed vertebrates 425 million years ago (mya) to the most recent
 antibody studied here, hominid IgE, just 2.5 mya, the homo-dimeric
 four-chain structure consisting of two identical heavy (H)-chains and two
 identical light (L)-chains has been preserved. In this conventionally
 drawn “Y-shaped” structure, two Fab arms interact with antigen, and the Fc
 region mediates effector functions such as complement activation and
 binding to cell surface receptors. While human IgM consists of either five
 or, less commonly, six of these
 H<sub>2</sub>L<sub>2</sub> units covalently linked
 in a (pseudo) hexagonal array), almost all other classes of antibody
 function as a single homo-dimer. The Fc regions of IgM and IgE both
 consist of six H-chain domains,
 (C<sub>H</sub>2-C<sub>H</sub>3-C<sub>H</sub>4)<sub>2</sub>, as does the evolutionary precursor to mammalian IgE (and IgG), namely avian IgY <sup>1,2,3</sup>, but in other classes of antibody, such as IgG, IgA and IgD, an extended “hinge” region replaces the C<sub>H</sub>2 domain; this permits greater flexibility between the Fab arms and the Fc region and is important for antigen binding.  However, conformational flexibility <em>within</em>the Fc regions of IgM and IgE is known to be critical for expressing their effector functions, but in different ways for these two isotypes, and structures from fully extended to acutely bent have been observed, as described below. Human IgM, free in serum in the absence of antigen, was shown in early studies to adopt a planar structure, with the Cm2, Cm3 and Cm4 domains in an extended conformation<sup>4,5</sup>. Both as pentamer and (more so) as hexamer, IgM is a potent activator of complement through the classical pathway, but only in the presence of antigen. Electron micrographs of IgM in the presence of excess antigen showed dislocation of the Fab arms from the central Fc disc to form “table” or “staple”-like structures, and these complexes activated complement <em>via</em> the classical pathway by binding of protein C1q<sup>4</sup>, the first sub-component of the complement cascade. Feinstein suggested that the C1q binding sites were obscured in the extended structure, and only became accessible in the dislocated, bent conformation<sup>77</sup>, a proposition supported by later small-angle X-ray scattering (SAXS) analysis and molecular modelling<sup>5</sup>.Whether the bend in the IgM-Fc was at the Cm1-Cm2 or Cm2-Cm3 junction was unclear, but the SAXS analysis suggested the latter<sup>5</sup>. Two recent cryo-EM studies of serum IgM (in which the Cm2 domains are visible), both alone<sup>6</sup> and in the presence of antigen and complement<sup>7</sup>, show that there is indeed a bend between the Cm2 and Cm3 domains, rather than between the Fabs and the IgM-Fc (<em>i.e.</em> not between Cm1 and Cm2). When IgM is bound to antigen, as in the latter structure, the (Cm2)<sub>2</sub> domain pair is bent by 100° from its fully extended position with respect to Cm3 and Cm4 in the flat disc<sup>7</sup>. Bending of IgM-Fc is an important factor for controlling the complement activity of this antibody. This mechanism is likely preserved through evolution as jawed fish have multimeric IgM, C1q and activate complement by the classical pathway<sup>8</sup>. Human IgE-Fc has the same three domain-pair composition (Ce2-Ce3-Ce4)<sub>2 </sub>(Fig.1A), but the crystal structure revealed an acutely bent conformation with the (Ce2)<sub>2</sub> domain pair bent back against the Ce3 domains and even making contact with Ce4<sup>9</sup>. This bent structure also predominates in solution <sup>10,11,12,13</sup>. The angle between the local two-fold axis of the (Ce2)<sub>2</sub> domain pair and that of the (Ce3-Ce4)<sub>2</sub> pair was 118° in the crystal structure<sup>8</sup> and this increased even further to 126° when the “high-affinity” receptor for IgE, FceRI, was bound between the two Ce3 domains, accompanied by an opening up of these domains<sup>14</sup>. In contrast, when the “low-affinity” IgE receptor FceRII/CD23 was bound to a site between the Ce3 and Ce4 domains, the bend decreased by 16° and the Ce3 domains adopted a more closed conformation<sup>15</sup>. As a consequence of these opposed conformational changes in the positions of the Ce3 domains relative to each other and to the Ce4 domains, binding of these two receptors is mutually exclusive – an allosteric effect<sup>16</sup>; when one site is accessible, the other is not, and <em>vice versa</em>, which is essential to prevent activation of an allergic reaction <em>via</em> FceRI, by multimeric CD23 in the absence of allergen. IgE-Fc can also adopt a fully extended conformation, which can be trapped and stabilised by anti-IgE-Fc antibody Fabs such as the aeFab<sup>17</sup>. However, for free IgE-Fc in solution, this extended conformation, to which FceRI binding is blocked sterically, may be only rarely populated<sup>17</sup>. Bending of IgE-Fc is thus important for modulation of receptor binding activity. IgY-Fc also has the same homo-dimeric composition as IgM-Fc and IgE-Fc, (Cu2-Cu3-Cu4)<sub>2 </sub> but only the crystal structure of the (Cu3-Cu4)<sub>2 </sub>domains is known<sup>18</sup> and not that of the (Cu2)<sub>2</sub> domain pair, which may be important in receptor and possible complement binding. This is the first structural analysis of the complete IgY-Fc molecule. One receptor, FcRY, is functionally similar to the mammalian transport receptor FcRn, and involved in transfer of maternal blood IgY to the yolk and then to the embryo<sup>19</sup>; it is known to bind to the Cu4 domains<sup>20</sup> and also to the Cu3/Cu4 interface<sup>19</sup>. There is also evidence from modelling of mutated IgY-Fc interactions with FcRY that changes in the angle between the Cu3 and Cu4 domains may occur<sup>19</sup>, akin to the conformational changes involving the Ce3 domains of IgE-Fc. Intriguingly, the crystal structure of IgY-Fc exhibits different Cu3-Cu4 angles in the two chains<sup>18</sup>, further indication that conformational change involving these domains may be possible. Another IgY receptor was identified in the chicken genome homologous to mammalian Fc receptors, FcR/L, but the protein has not been prepared<sup>21</sup>. A further two receptors, ggFcR<sup>22</sup> (on chromosome 20) and CHIR-AB1<sup>23</sup> (with a gene in the Leukocyte Receptor Complex) are known and bind at sites homologous to firstly, the FceR1 site on IgE-Fc between the Cu3 domains<sup>22</sup>, and secondly, the FcaR1 site on IgA-Fc, close to the CD23 binding site on IgE-Fc between the Cu3 and Cu4 domains<sup>24</sup> respectively. In chickens, which have only the IgM, IgA and IgY isotypes, the latter performs the functions of both human IgG<sup>25 </sup>and IgE<sup>26,27</sup>, but the functions of the two receptors ggFcR and CHIR-AB1, and whether there is any allosteric communication as in IgE, remains to be investigated. Unlike IgM and IgE, the disposition of the (Cu2)<sub>2</sub> domain pair of IgY-Fc in solution (or crystal) has not previously been studied. There is a large gap between the appearance of  IgY 310 mya and hominid IgE 2.5 mya, but this is bridged by the monotreme platypus IgE which appeared 166 mya<sup>28</sup>. We have used extant proteins due to their availability and the better, although incomplete understanding of their function. Our justification for doing so is that the domain structure of all three antibody isotypes has remained constant through evolution, and also that for IgM, although its polymeric state has varied, its complement activation function has been conserved. In this paper, SEC-SAXS (Size Exclusion Chromatography-SAXS) was employed to study the solution conformations of the Fc regions of these four antibodies. In the case of IgM-Fc, the homo-dimeric subunit of the human pentamer or hexamer was used, to permit comparison with the other antibody Fc domains. SEC-SAXS measures the X-ray scattering as the protein is eluted from a size exclusion column, to better resolve monomeric from aggregated material. The scattering data not only permit analysis of the average conformation of each protein in solution, but also detect whether more than one, <em>i.e. </em>an ensemble, of different conformations is present. We track the evolution of conformational variability and function through this series of antibodies. <strong>Materials and Methods</strong> <em>Protein preparation</em> All four Fc proteins, human IgE-Fc (huIgE-Fc), platypus IgE-Fc (plIgE-Fc), chicken IgY-Fc (chIgY-Fc) and human IgM-Fc (huIgM-Fc) are glycosylated. huIgE-Fc has three N-glycosylation sites at asparagine residues 265, 371 and 397. The latter residue plays a structural role in the Fc, located “internally” between the two Ce3 domains, and was retained, whereas N265 and N371 were mutated to glutamine. In plIgE-Fc, chIgY-Fc and huIgM-Fc, the homologous site to 397 was retained, but other sites identified in biochemical studies or using Net-Gly<sup>29</sup> were mutated to glutamine. huIgE-Fc (N265Q, N371Q) secreted from a stable NS-0 cell line was purified from tissue culture supernatant by cation exchange chromatography<sup>17</sup> Supernatant was buffer-exchanged into 50 mM sodium acetate pH 6.0, 75 mM NaCl and loaded onto a SPHP cation-exchange column (GE Healthcare). huIgE-Fc (N265Q, N371Q) was eluted with a 10 × column volume gradient into 50 mM sodium acetate, pH 6.0, 1 M NaCl. Eluted fractions were pooled, concentrated and further purified by SEC on a Superdex G200 column (GE Healthcare) in PBS, pH 7.4. The protein sequences of the other three Fc fragments, were chosen to align with the N- and C- termini of the huIgE-Fc. C414 in huIgM-Fc, which mediates pentamer or hexamer formation, was mutated to serine. Sequences were pipe-cloned<sup>30</sup> into pcDNA5, preceded by a mouse kappa light chain leader sequence and followed by bases coding for six histidine residues, to facilitate secretion of the Fc proteins by expression in HEK293F cells, and purification, respectively. Cell supernatants were purified on a Ni-NTA column (Thermo Fisher Scientific) followed by SEC chromatography in tris-buffered saline with azide pH 7.5 using a Superdex G200 column (GE Healthcare). Protein purity was assessed using reduced 5 mg aliquots on a 10% SDS-PAGE gel, and the molecular mass estimated by SEC-MALLS (Size Exclusion Chromatography-Multi-angle Laser Light Scattering), which combines multi-angle light scattering with size-exclusion chromatography to estimate a shape-independent molecular mass. Glycosylation at the conserved N-glycosylation site was checked by comparison of the molecular mass of 2.5 mg protein samples incubated with or without PNGaseF (NEB P0704S) according to the manufacturer's instructions. The gel was calibrated with molecular mass markers by plotting log molecular mass <em>vs</em>. migration<sup>31</sup>. <em>SAXS data collection</em> Data were collected at the Diamond Light Source (BL21). Each of the four proteins was prepared twice. Data were collected for the first set of four proteins by SEC-SAXS with a Shodex KW403 column, and for the second set with a Superdex G200 Increase column. <em>FPMod models</em>  The conformational space occupied by all four proteins was sampled using models of huIgE-Fc and huIgM-Fc generated by FPMod<sup>32</sup>. Models for plIgE-Fc and huIgE-Fc were generated from the acutely bent<sup>9</sup> (PDB 1O0V) and fully extended<sup>17</sup> (PDB 4J4P) crystal structures of huIgE-Fc, arranged as two rigid bodies, (Ce2)<sub>2</sub> and (Ce3-Ce4)<sub>2</sub>, joined in one of the chains by the flexible linker of three amino acids, DSN. Models for huIgM-Fc and chIgY-Fc were generated from a model of fully extended huIgM-Fc (using the sequence of huIgM-Fc with the crystal structure of fully extended huIgE-Fc as template), again arranged as two rigid bodies and using a flexible linker of 7 amino acids, VPDQDTA (from IgM-Fc) in one of the chains. These seven residues are not visible in the cryo-EM structure of full-length human IgM<sup>6</sup> (PDB 8ADY) and so they are probably flexible. The preceding cysteine forms a disulphide bond between the heavy chains<sup>33,34</sup>. <em>Ensemble selection</em> The intensity plots from the first set of protein preparations were used as input to a non-negative linear least-squares algorithm, NNLSJOE<sup>Footnote, 35,36</sup>, in EOM 3.0. The pool of models (of huIgE-Fc for the huIgE-Fc and plIgE-Fc intensity plots and huIgM-Fc for huIgM-Fc and chIgY-Fc intensity plots) were analysed by NNLSJOE, resulting in a set of several models for each protein which best fitted the corresponding experimental intensity plot. The number of runs<sup>35</sup> was 100. Fits with values of χ<sup>2</sup>&lt;2 were accepted. Each set of models was distributed according to Rg values into four bins representing acutely bent, partially bent and fully extended protein conformations. <strong>Results</strong> <em>Protein preparation</em> The purity of the proteins as judged by SDS PAGE; the molecular masses, estimated by SEC-MALLS, of huIgM-Fc, chIgY-Fc, plIgE-Fc and huIgE-Fc were 72, 79, 80 and 73 kD respectively. Reduced heavy chains of each protein can be seen with a molecular mass of just &gt;35kD consistent with their calculated sizes of 36 to 40 kD. Evidence for glycosylation at the conserved N-glycosylation site was seen using  SDS-PAGE, by incubation with (+) or without (-) PNGaseF. Each protein shows a molecular mass difference of several kD; crystal structures of huIgE-Fc (PDB 1O0V and PDB 2WQR), have about 2 kD of carbohydrate on both chains together<sup>9,14</sup>. <em>SAXS analysis</em> SAXS analysis permits calculation of the radius of gyration, Rg, a measure of the distribution of mass about the centre of gravity of the protein; more extended conformations have larger Rg values than compact structures. The full X-ray scattering curves (intensity as a function of scattering angle) are sensitive to details of protein shape, and as described in the following  section, these were analysed using a non-negative linear least-squares algorithm<sup> </sup>to choose from a pool of models those which best fit the data, and determine whether one or a number of different conformations was present in solution. Two separate sets of preparations of the four Fc proteins were used for collection of SAXS data and calculation of Rg values.Tthe Rg values are similar for the two sets of preparations. Data were imported into Chromixs<sup>37</sup> in ATSAS 2.8<sup>38</sup> for buffer subtraction and generation of elution profiles and into Scåtter<sup>39 </sup>for subsequent generation of intensity, Guinier, P(r) and dimensionless Kratky plots .Some aggregation was evident in huIgM-Fc, chIgY-Fc and plIgE-Fc Consequently, the frames 253-262, 270-278, 248-258, and 278-291 for the four proteins huIgM-Fc, chIgY-Fc, plIgE-Fc and huIgE-Fc respectively, were used to generate intensity plots. Calculated average Rg values for each of the frames (1 sec. exposure) were used to assess the range of average Rg values across the part of the peak used. Intensity plots for each of the four proteins were analysed in Scatter<sup>39</sup>. Average Rg values in reciprocal space were estimated using a Guinier plot (ln I <em>vs</em>. q<sup>2</sup> gives a straight line with slope –Rg/3) (Fig. 3B) with qRg values below 1.3 and residuals symmetric about zero<sup>40</sup>. The lower average Rg values for huIgE-Fc and plIgE-Fc (29.7Å and 30.4Å), compared with chIgY-Fc and huIgM-Fc (35.6Å and 36.8Å) indicate that the IgE-Fc molecules adopt a more compact structure on average than chIgY-Fc and huIgM-Fc, which adopt, on average, more extended structures. Plots of q vs. log(I) with q&lt;0.1 emphasize the difference between the more extended (huIgM-Fc and chIgY-Fc) and more compact (plIgE-Fc and huIgE-Fc) configurations at q ~ 0.05 Å<sup>-1</sup>. It is striking that the range of average Rg values over the frames used for each protein analysis is significantly smaller for plIgE-Fc and huIgE-Fc than for huIgM-Fc and chIgY-Fc This most likely reflects the conclusion reached from the analysis presented in the following section, that both IgE-Fc molecules predominantly adopt an acutely bent, compact conformation and thus present a more structurally homogeneous population of molecules than chIgY-Fc and huIgM-Fc, which adopt a greater range of conformations from bent to extended. These different conformations may be differentially retarded on the column, and consequently the average Rg values vary to a greater extent across the peak for chIgY-Fc and huIgM-Fc. These ranges of average Rg values for each protein derived for the first preparation also encompass the range of values derived from the second preparation. While we cannot place statistical significance estimates on these Rg values, given only two experimental replicates, those Rg values derived from the second preparation add confidence in the values and ranges for the first The maximum dimension of each protein, D<sub>max </sub>, was estimated from the P(r) function This shows that the maximum length of huIgM-Fc and chIgY-Fc (~110Å and ~105Å respectively) are greater than that of plIgE-Fc and huIgE-Fc (~90Å). (Values for Preparation 2 are in good agreement; huIgM-Fc: ~116Å, chIgY-Fc: ~104Å, plIgE-Fc: ~94Å and huIgE-Fc: ~98Å).  A dimensionless Kratky plot (where (qRg)<sup>2 </sup>I(q)/I(0) is plotted against qRg) was used to assess the globularity of the proteins huIgE-Fc and plIgE-Fc appear globular with a characteristic maximum close to y = 1.1, x = √3, whereas huIgM-Fc and chIgY-Fc have a peak at higher x values suggesting a more extended protein<sup>40</sup>. Although the experimental Rg values discussed above are average values for each protein, it is instructive to compare them with the Rg values calculated for two crystal structures of huIgE-Fc discussed earlier in the Introduction. One of these is free huIgE-Fc<sup>9</sup> (PDB 1O0V), which is acutely bent between the Ce2 and Ce3 domains and adopts a very compact structure; the other is that of huIgE-Fc stabilized in a complex in a fully extended conformation<sup>17</sup> (PDB 4J4P). The calculated Rg value for the former is 28.8Å, and for the latter, 35.6Å (Supplementary Table 4). The experimental, average Rg values of both huIgE-Fc (29.7Å) and plIgE-Fc (30.4Å) are close to the value for the acutely bent huIgE-Fc structure. In contrast, the experimental, average Rg values of huIgM-Fc (36.8Å) and chIgY-Fc (35.6Å) are close to the value for the fully extended huIgE-Fc structure. This is also true for the average Rg values derived from the second protein preparation. <em>Models for interpretation of the SAXS data</em> Ensembles of models were generated which maximize the fit of the theoretical to the experimental intensity plots, using a pool of either huIgE-Fc models or huIgM-Fc models (see Materials and Methods). The ensembles chosen by NNLSJOE consisted of a minimum of 2 and a maximum of 7 models, and thus always included structures other than the predominant acutely bent or fully extended configuration. Figure 4 shows the Rg values of each of the models chosen and their relative contribution to fit the scattering data for each of the four proteins. For huIgM-Fc, the SAXS results show that no acutely bent structures are present. There are however clusters of both fully extended and partially bent structures. The former constitute 64% of the structures (4 models with Rg values 38.0Å (15%), 38.1Å (27%), 38.2Å (19%) and 38.5Å (3%)), the latter 36% of the structures (3 models with Rg values 32.8Å (14%), 32.8Å (6%) and 33.3Å (16%)). Since bending of the IgM-Fc is proposed to expose the binding site for C1q<sup>4,5</sup> the question arises: which of the conformations observed are capable of binding C1q? Complement binding residues in IgM-Fc have been identified by site-directed mutagenesis<sup>41</sup>, and they are inaccessible to C1q in the fully extended IgM-Fc molecule. However, the complex of C1q bound to the homologous region of human IgG-Fc in the cryo-EM model<sup>42</sup> (PDB 6FCZ) can be used to assess whether C1q might bind to the partially bent structures of huIgM-Fc observed in the present study. The (Cg2-Cg3)<sub>2</sub> domains of IgG-Fc in the C1q complex (PDB 6FCZ) were superposed on each of the seven models of huIgM-Fc selected by the algorithm NNLSJOE, using matchmaker from ChimeraX, and in the four fully extended models representing 64% of the material, the Cm2 domains clashed sterically with the C1q “head”. The other three, partially bent  models, did not clash. The fact that this implies that 36% of the material adopts conformations sufficiently bent to allow access to C1q, is discussed below. Whilst for huIgM-Fc there are no acutely bent models represented, chIgY-Fc contains material which is acutely bent, partially bent and fully extended These constitute 31%, 34% and 35% of the structures respectively. In contrast, both plIgE and huIgE are predominantly acutely bent, although both indicate the presence of some partially bent material For plIgE-Fc, the acutely bent structure constitutes 67% of the material (Rg 28.7Å), with 33% partially bent (Rg 31.4Å), while for huIgE-Fc, 84% (Rg 29.2Å) is acutely bent and 16% (Rg 35Å) is partially bent.  Neither plIgE-Fc nor huIgE-Fc shows evidence of any fully extended material. The four proteins form a series in which the proportion of acutely bent material increases: IgM-Fc &lt; IgY-Fc &lt; plIgE-Fc &lt; huIgE-Fc. This follows their order of appearance in evolution. The ensemble of conformations for IgY-Fc from acutely bent to fully extended, rather than the predominantly acutely bent conformation of IgE-Fc and predominantly fully extended conformation of IgM-Fc, reflects IgY’s position as an evolutionary intermediate between IgM and IgE. These conclusions are supported by analysis using NNLSJOE of the data from preparation 2. Although there are some dfferences in the proportions of partially bent conformations, there is very good agreement for the proportions of acutely bent and fully extended conformations. The proportion of acutely bent material increases IgM-Fc &lt; IgY-Fc &lt; plIgE-Fc &lt; huIgE-Fc (0%, 31%, 77% and 83% for preparation 2, <em>cf.</em> 0%, 31%, 67% and 84% for preparation 1); huIgM-Fc displays no acutely bent conformations and is predominantly fully extended (70% for preparation 2, <em>cf.</em> 64% for preparation 1); huIgE-Fc and plgE-Fc are predominantly acutely bent (83% and 77% respectively for preparation 2, <em>cf. </em>84% and 67% for preparation 1) and display no fully extended conformations; chIgY-Fc displays both acutely bent and fully extended conformations (31% and 69% respectively for preparation 2, <em>cf.</em> 31% and 35% for preparation 1). <strong>Discussion</strong> Homo-dimeric Fc regions, (C<sub>H</sub>2-C<sub>H</sub>3-C<sub>H</sub>4)<sub>2</sub>, of the four antibodies, human IgM, human and platypus IgE, and chicken IgY were prepared, with glycosylation at the conserved “internal” site in C<sub>H</sub>3, for analysis of their solution conformation. SEC-SAXS was employed, and the intensity data were analysed using a non-negative linear least-squares algorithm to choose from a pool of models to fit the data and determine whether one or a number of different conformations was present in solution. The analysis suggests the presence of more than one conformation in each case. The crystal structure of huIgE-Fc reveals an acutely bent conformation<sup>9 </sup>which appears also to predominate in solution, as determined by earlier SAXS<sup>11,13</sup> and FRET<sup>10,12</sup> analyses. The energy landscape for IgE-Fc has been explored by molecular dynamics simulations, and an estimate of the energy difference between the acutely bent and fully extended conformations (~20 kJ/mol) suggests that there is little extended IgE-Fc in solution<sup>17. </sup>This was confirmed in the present study, with 84% of the material estimated to be in an acutely bent conformation. In contrast, for huIgM-Fc, the SAXS results show that no acutely bent structures are present, but there are both fully extended and partially bent structures (Fig. 4). We have shown, by comparison with the cryo-EM model of C1q “head” bound to the homologous IgG-Fc (PDB 6FCZ) as described above that in 36% of these structures the C1q binding site is accessible. However, free pentameric IgM does not bind C1q or activate complement, suggesting that formation of the pentamer (or hexamer), may affect the conformational diversity in IgM-Fc. Although pentameric IgM<sup>6</sup> and IgM-Fc<sup>43,44,45</sup> cryo-EM structures imply that flexibility and bending at the Cm2-Cm3 interface can occur, even in the absence of antigen, the extent of this bending is not known. Other factors such as glycosylation may also affect the ability of IgM-Fc to bend and activate complement. A recent study showed that in severe COVID-19 patients’ sera, increased glycosylation in IgM-Fc correlated with increased IgM-dependent complement deposition<sup>46</sup>. We note that in the recently determined structures of the IgM B-cell receptor<sup>33,34,47</sup>, the Fc region adopts an extended conformation, perhaps stabilized by the accessory Iga and Igb molecules. IgE-Fc from platypus, evolutionarily the the most primitive of extant mammals, was included in this study to offer an evolutionary intermediate between chicken IgY-Fc and human IgE-Fc. In fact, the profile of conformations seen for plIgE-Fc is very similar to that of huIgE-Fc, dominated by an acutely bent conformation (67%). CD23 has been annotated in the platypus genome<sup>48</sup> and fragments of an FceR1a homologue have been identified with Blast<sup>48</sup>. However, key residues in the huIgE-Fc binding site for CD23<sup>15,16</sup> are not conserved in plIgE-Fc, and binding of platypus FceR1 to plIgE-Fc has never been demonstrated, nor have there been reports of an allergic reaction, but an individual of the only other extant monotreme, echidna, was found to be allergic to its main source of food, namely ants<sup>49</sup>. The presence of IgE, CD23 and possibly FceR1a in the platypus genome suggest that the system seen in humans<sup>50</sup> had already evolved to combat helminths, which do infect monotremes and marsupials<sup>51</sup>. Both acutely bent and fully extended structures occur in the ensemble for chIgY-Fc, together with intermediate conformations, clearly different from the distributions seen for either IgM-Fc or IgE-Fc. The classical complement pathway has been found in the chicken<sup>52</sup> but its activation could be due to IgY or IgM. There are no structural or mutational data to indicate how chicken IgY might interact with chicken complement, but two out of the three important residues in both the human IgG and IgM interaction with human complement, P329 and P331, align with prolines in IgY. Chicken IgY only exists as a homo-dimer (in contrast to the pentameric or hexameric IgM) and the SAXS results show that 31% of it (as judged by performing the same superposition of the IgG-Fc/C1q complex<sup>42</sup> (PDB 6FCZ) as described above for IgM-Fc) is bent sufficiently to expose the complement binding site, if it exists; the disposition of the (Cu2)<sub>2</sub> pair is therefore unlikely to affect activation. A range of conformations for chicken IgY-Fc and the putative C1q binding site is possible.. If IgY is able to activate chicken complement, the formation of polymeric structures upon antigen binding is likely to be the control mechanism, as it is with IgG<sup>53,54</sup>, because enough of the IgY-Fc is sufficiently bent to allow C1q to bind. Although chickens do experience anaphylaxis<sup>26</sup>,<sup> </sup>there is no evidence that IgY and a receptor are involved. On the other hand, it is known that IgY has some functions analogous to those of both human IgG and IgE, which may involve the identified receptors ggFcR<sup>22</sup> and CHIR-AB1<sup>23</sup>, which bind to IgY-Fc at similar locations, respectively, to FceRI and CD23 on IgE-Fc<sup>22,24 </sup>. The presence of more than one bent conformation suggests that, like huIgE-Fc, different dispositions of the (Cu2)<sub>2</sub> domain pair may correspond to the binding of different chicken receptors, and changes in the Cu3-Cu4 angle may also occur<sup>18,19</sup>, as described in the Introduction, similar to those that are associated with the open and closed conformations of the Ce3 domains in IgE-Fc<sup>14,15</sup>. Allostery in relation to IgY receptor binding might therefore be possible as it is for IgE. We have studied and compared the solution structures of human IgM-Fc, platypus and human IgE-Fc and chicken IgY-Fc, the latter being the first structural study of the complete Fc region of this isotype. While IgM-Fc is principally fully extended in solution, with no acutely bent conformations, IgE-Fc is principally acutely bent in solution, with no fully extended conformations. In contrast to both IgM-Fc and IgE-Fc, IgY-Fc displays an ensemble of conformations from acutely bent to fully extended, reflecting IgY’s position as an evolutionary intermediate between IgM and IgE. Indeed, the proportion of acutely bent conformation, IgM-Fc &lt; IgY-Fc &lt; plIgE-Fc &lt; huIgE-Fc, follows their order of appearance in evolution. The solution structure of human IgM-Fc (which was already present 425 mya in jawed fish) is consistent with IgM using the bend between the (Cm2)<sub>2</sub> domain pair and (Cm3-Cm4)<sub>2</sub>,influenced by glycosylation and/or multimerization, to control complement activation upon antigen binding. Hominid IgE (which only appeared 2.5 mya), does not bind complement, but the bend between (Ce2)<sub>2</sub> and (Ce3-Ce4)<sub>2</sub> is associated with the mutually exclusive, allosteric binding of two receptors, FceRI and CD23. This function probably also exists in the monotremes, platypus and echidna (which first appeared 166 mya). The solution structure of IgY-Fc (which appeared 310 mya in tetrapods) suggests that it has lost the facility to control complement binding using the (Cu2)<sub>2</sub> domain pair, and may have already co-opted that binding site for Fc receptor binding, opening up the possibility of allosteric control of receptor binding as seen in human IgE. In summary, IgY occupies an intermediate position in the evolution of antibody structure from the most primitive IgM to the most recent IgE. This is the first structural analysis of the complete IgY-Fc, allowing direct comparison with IgM-Fc and IgE-Fc; we find that IgY-Fc displays a unique ensemble of conformations in solution, distinct from IgM-Fc and IgE-Fc but displaying features of both. 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description: # Functional evolution of antibody Fc domains: SAXS plots and models
 [https://doi.org/10.5061/dryad.6q573n66m](https://doi.org/10.5061/dryad.6q573n66m) ## Description of the data and file structure 1-dimensional x-ray scattering plots were provided by Diamond: Prep1:08-04-16, huM1, chY1, plE1, huE1. Prep2:05-08-16, huM2, chY2, plE2, huE2. The circular scattering pattern was averaged to create the plots. Data was collected for 1 second every 3 seconds as each protein was eluted from the SEC column to generate 500-600 files. Each file has 3 columns: q (~0 to 0.4) or momentum transfer in reciprocal space, giving size and shape information about the proteins, I or Intensity of scattered x-rays, error (in intensity). Files were dropped into Chromixs (ATSAS2.8) and Rg values calculated across the peak of the elution profile. Frames were chosen for further processing, avoiding aggregated protein. The intensity of scattered x-rays from the chosen frames were averaged and dropped into Scatter (v.3a). Files were truncated at low q to achieve linear Guinier plots and at high q to obtain smooth P(r) plots with a single intersection of the x axis. Truncated .dat files can be converted to text files (which are not necessarily accepted by SAXS software). Pdb models: Mmodels for use with huIgM-Fc and chIgY-Fc and Emodels for use with plIgY-Fc and huIgE-Fc were generated from models (extended M) or pdb structures (E) of both bent and extended huIgE-Fc. For use in FPModGUI/ conformation sample, Fc chain A starts after the linker DSN. Chain B consists of Chain A before DSN joined to the whole of chain B. To run the programme, chain ID is B. Terminal to append to is N, huge sequence is fasta files of both A and B and exact sequence is DSN. 1000 models of huIgE-Fc were generated starting from the bent structure 10OVpdb and 1000 models from the extended structure 4J4Ppdb. 1000 models of IgM-Fc were generated. Formfactors for each set of models were generated in EOM3(ATSAS2.8) creating intensities files and size_statistics files for use in NNLSJOE. For each prep, NNLSJOE chose an ensemble of models giving rise to a SAXS intensity plot which best fitted the experimental plot. Chosen models are shown in NNLSJOE1 and NNLSJOE2.  Further detail is contained in DOI: 10.3389/fimmu.2024.1389494.