Thermal sensitivity and flexibility of the Cϵ3 domains in immunoglobulin E

The C  3 domains of IgE are the most susceptible to thermally induced unfolding, determined by differential scanning fluorimetry. The C  3 domains are responsible for the characteristically low melting temperature of IgE. The C  3 domains exhibit the greatest intrinsic flexibility. The quaternary structural diversity of the C  3 domains is compared across all (34) known structures using a simplified single parameter. Human IgE-Fc and Fc  3-4 domain structures are determined at the highest resolutions yet reported (1.75Å and 2.0Å respectively)


Introduction
IgE plays a central role in the molecular and cellular mechanisms of allergy, and is a validated therapeutic target in the development of new approaches to treat allergic diseases such as asthma.It exerts its effects through its two principal receptors, FcRI on mast cells, basophils and antigen-presenting cells, and CD23/FcRII on B cells.
The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both contribute to allergen presentation to the immune system [1,2].
IgE causes long term sensitisation of mast cells due to the uniquely slow off-rate for the antibody/receptor complex (k off = 1.9 x 10 -4 s -1 ), and this is reproduced with the IgE-Fc fragment containing the dimer of C2, C3 and C4 domains [3].The FcRI binding site comprises two sub-sites, one on each C3 domain [4,5], and although the C2 domains are not directly involved, they contribute to this slow off-rate since the Fc3-4 fragment, lacking these domains, has a somewhat faster off-rate (k off = 3.2 x 10 -3 s -1 ) [3].The overall affinities of IgE-Fc and Fc3-4 for FcRI are, however, comparable and high (K a ≈ 10 10 M -1 ).The CD23 binding site lies principally within the C3 domain but with a small contribution from C4 [6]; the affinity of both IgE-Fc and Fc3-4 for CD23 is lower (K a ≈ 10 7 M -1 ), with one binding site in each chain.
The C3 domain is thus the critical domain for receptor binding.
When expressed alone, and in contrast to C2 and C4, the C3 domain displays a poorly ordered, "molten globule" structure [7,8,9,10].However, this isolated domain binds, albeit with lower affinity, to sFcRI [11,7], in the process becoming more ordered [8,9].This is also the domain to which N-linked carbohydrate is attached, at a site that is conserved across other immunoglobulin isotypes; in IgE the glycosylation is of the high-mannose type [12,13,14,15].
There are now several crystal structures of both IgE-Fc and Fc3-4 alone and in complex with soluble IgE-binding extracellular domains of FcRI (sFcRI), CD23 (sCD23) and various inhibitors.The first crystal structure was of Fc3-4 bound to sFcRI (1F6A) [4], and this was followed by that of the free Fc3-4 (1FP5) [16].

A C C E P T E D M A N U S C R I P T 5
The structure of IgE-Fc revealed the organization of the C2 domains for the first time (1O0V) [17], and caused much surprise when they were observed bent back onto the Fc3-4 domains in an asymmetric manner.Three new crystal forms of Fc3-4 were then determined (3H9Y, 3H9Z, 3HA0) [18], which revealed conformational flexibility between the structures.IgE-Fc bound to sFcRI (2Y7Q) was determined along with a higher resolution structure of the free IgE-Fc (2WQR) [5].The structure of Fc3-4 with an engineered disulphide bond, trapping Fc3-4 in an artificially closed, non-receptor binding conformation was determined (4GT7) [19] and the structure of the same constrained form of Fc3-4 was determined bound by two DARPin (designed ankyrin repeat protein) molecules (4GRG) [20].The structure of Fc3-4 bound to calcium-free sCD23 was then determined (4EZM) [6] and subsequently the calcium-bound structure was also solved (4GKO) [21], as well as another crystal form of Fc3-4 bound to sCD23 (4KI1) [22].IgE-Fc bound by two anti-IgE Fabs (aFabs) (4J4P) [23] unexpectedly captured the Fc in a fully extended conformation.The crystal structure of Fc3-4 bound by the neutralizing antibody Fab MEDI4212 was also determined (5ANM) [24].The complexes of two anti-IgE omalizumab Fabs bound to the artificially constrained Fc3-4 (5HYS) [25] and to the native IgE-Fc (5G64) [26] were recently reported.The structure of the asymmetric complex of IgE-Fc bound to a single sCD23 molecule was also recently solved (5LGK) [27].
In FcRI-bound structures, the C3 domains move apart into an "open" conformation to accommodate receptor binding, compared with the uncomplexed fragments.
However, binding to CD23 between the C3 and C4 domains causes the C3 domains to adopt a more "closed" conformation, and in this way the binding to the two receptors, while occurring at distant sites at either end of the C3 domains, is mutually exclusive [6,28].
The thermal lability of IgE has been recognised for a long time.Even before the recognition of IgE as an antibody, the loss of "reaginic" activity in atopic sera by heating to 56C was reported [29], and shortly after the discovery of the IgE antibody class, this unusually low thermal susceptibility compared with IgG (for which T m values are typically ~72C), was confirmed [30].

ACCEPTED MANUSCRIPT
A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT
A C C E P T E D M A N U S C R I P T 7

Protein preparation and crystallisation
IgE-Fc and Fc3-4 were prepared as reported earlier [31,6], and were a kind gift from Dr. A.J. Henry (UCB Pharma, Slough, UK).Crystals were grown at 18°C using the sitting drop vapour diffusion method.Fc3-4 crystals in the P1 and P2

ACCEPTED MANUSCRIPT
A C C E P T E D M A N U S C R I P T 8 were collected using a Pilatus 6M-F detector (1800 images, 0.2 oscillation, 0.2 s exposure, λ 0.92819Å).Data were integrated with DIALS using the Xia2 package [32] and scaled with AIMLESS [33] from the CCP4 suite [34].
The number of molecules in each asymmetric unit (Table 1) was correctly predicted using Matthews probability analysis [35,32].All structures were solved by molecular replacement with PHASER [37] using the Fc3-4 structures, PDB entries 3HA0, 3H9Y and 3H9Z [18], as search models for the P1 and P2 1 'large' and P2 1 'small' structures, respectively.For the IgE-Fc structure, PDB entry 2WQR [5]   and structure 3H9Y chain A, B and C), the measurement was not taken.

B-factor analyses of structures
All chains of the four new structures presented here, together with all published Fc3-4 and IgE-Fc structures, were analysed.To compare B-factors derived from different structure determinations, each was normalised by division by the average B-factor for the whole C3-4 chain (for comparison of C3 and C4) or C3 domain (for comparison of the C4-distal region and C4-proximal region).The C3/C4 boundary was defined as residues 437/438; the C4-distal region of C3 was defined as residues 336-339, 359-372, 387-400 and 419-431; the C4-proximal region of C3 was defined as residues 340-358, 373-386, 401-418 and 432-437.

Differential scanning fluorimetry
Thermal stability assays were carried out according to a published protocol [41] using the Stratagene Mx3005p RT-PCR instrument.Sypro orange was used as fluorescent dye (Life Technologies) with filters at 492nm (excitation) and 610nm (emission).The concentration for both proteins was 5M.Each sample was measured in duplicate for Fc3-4 and in triplicate for IgE-Fc.Positive controls were performed with lysozyme (data not shown) and negative controls as follows: protein with no Sypro orange, Sypro orange with buffer, and buffer alone (data not shown).Melting temperatures were taken as the maxima of the first derivative calculated using Graphpad Prism version 7 (Table 2).

Mass spectrometry
The two protein samples, either IgE-Fc or Fc3-4, were dialysed into a solution of H 2 O, 10% acetonitrile and 0.1% formic acid prior to mass spectrometric analyses.
Samples at concentrations between 8μg/mL and 80μg/mL were directly infused at 25μL/minute into a Bruker MaXis ultrahigh-resolution electrospray ionization quadropole time-of-flight mass spectrometer.The multiple charge states were deconvoluted using the manufacturer's maximum entropy algorithm (Bruker Daltonics).Due to glycosylation heterogeneity, a series of masses were observed with well-defined mass differences of 162 Da, corresponding to variations in the number of mannose residues present in the sample [12].For IgE-Fc, the derived masses were consistent with intact protein with a single N-linked glycosylation site per chain, with two GlcNAc residues and 1-7 mannose residues.For Fc3-4, the derived masses were consistent with intact protein with a single N-linked glycosylation site per chain, containing two GlcNAc residues and 5-9 mannose residues.Both protein samples also show evidence of partial loss of their C-terminal lysine residue, a common modification observed in recombinantly expressed antibodies [42].

Overall structure
The new crystal forms of Fc3-4, P1 and P2 1 'small' contain three molecules (six chains) in the asymmetric unit (solved at 2.2Å resolution), and one molecule (two chains) in the asymmetric unit (solved at 2.26Å) respectively.They therefore offer four additional and unique packing environments for analysis of the intrinsic flexibility of this molecule (Figs.1A and 1C).The other crystal form, P2 1 'large', with two molecules (four chains) in the asymmetric unit, has been reported earlier [18] but is now solved at 2.0Å resolution, the highest yet reported for any Fc3-4 structure (Fig. 1B).The IgE-Fc structure, space group P2 1 2 1 2, with one molecule (two chains) in the asymmetric unit has been determined to 1.75Å resolution (Fig. 1D), which is the highest reported for this structure.The structure of the carbohydrate, disposition of domains compared with other IgE-Fc and Fc3-4 structures, and relative flexibility within and between domains, will be discussed in the following sections.
A remarkable feature of the carbohydrate attached to chain D in the Fc3-4 P2 1 'large' crystal form and chain F in the Fc3-4 P1 crystal form is that while the terminal units are well defined, there is poor (or absent) electron density at a contour level of 1 in the 2FoFc map for the first GlcNAc (P2 1 'large') or first two GlcNAc units (P1), as well as for the covalent connection between these and Asn394 (Figs. 3B   and 3D).This covalent connection is well defined however in chain D of the Fc3-4 P2 1 'large' structure, and chain E of the Fc3-4 P1 structure (Figs.3A and 3C).
The terminal units make clearly defined contacts with residues of the C3 and C4 In IgG-Fc, complex-type carbohydrate chains are attached at the structurally homologous Asn297 in C2, but in IgG contact is made only with the C2 domains, and never C3.In IgE, the more extensive high-mannose structures have the capability of reaching the C4 domains, and the Fc3-4 P2 1 'large' structure reported here shows how the terminal carbohydrate units can be stabilised and contribute to linking, non-covalently, the C3 and C4 domains in IgE, even when the Asn394 loop to which it is covalently connected is relatively disordered.

Quaternary structural comparisons
In their earlier analysis of the quaternary structural variation in IgE-Fc and Fc3-4, Wurzburg and Jardetzky defined two motions of the C3 domains relative to each other, an "opening/closing" and a "swinging" [18].The C4 domain pair is virtually identical in all structures and provides a reference point for comparing the disposition of the C3 domains.These authors defined two distance measurements: C of residue 394 of one chain (X) to C497 of the other chain (Y) for the "opening/closing", and C336(X) to C336(Y) for the "swinging".These were displayed as a 2d plot [18].
As these are in fact one concerted movement, with more "closed" structures showing less "swing" and more "open" structure showing more "swing" we propose combining both components with a single measurement of the angle defined by C337(X)-C497(X)-C337(Y) (Fig. 5), using the descriptors "open" and "closed".
Residue 337 was selected as it is located within the C3 domain, away from the C2-C3 domain linker which undergoes a conformational change when IgE-Fc unbends [23].
We have compared all available IgE-Fc and Fc3-4 structures (now 34 molecules, compared with only 10 in the earlier analysis [18]); the single parameter clearly describes the wide range of open and closed C3 conformations (Fig. 5).The domain contact [23].When this occurs, the C3 domains adopt a quaternary structure that is incompatible with FcRI binding; the quaternary structure lies between the two extremes (Fig. 5).A similar phenomenon was observed for Fc3-4 bound to another anti-IgE Fab, MEDI4212 [24].The artificially constrained Fc3-4 structures and complexes with DARPin and omalizumab [20,19,25], are clearly more closed (Fig. 5).The C3 domains in this Fc3-4 construct are, however, tethered by an artificial disulphide bond that prevents any relative movement.In contrast, IgE-Fc bound to omalizumab exhibits the most open conformation seen, and is also incompatible with FcRI binding [26]; IgE-Fc is partially bent in this complex.It is also clear from this comparison that despite the 26 independent chain conformations observed in free Fc3-4 (and the single uncomplexed IgE-Fc structure), none is sufficiently open to permit FcRI binding; engagement of receptor must occur at one sub-site before full opening occurs.In contrast, many of the free Fc3-4 structures adopt conformations that are receptive for CD23 binding.

Flexibility of the C3 domains
The new structures show disorder in the loops and adjoining -strands of the region of C3 most distant from the C4 domains.(The two halves of the C3 domain will here be referred to as the C4-proximal and C4-distal regions; for definition by residue number, refer to Materials and methods).While this has been seen in other Fc3-4 (and also some IgG-Fc structures [41]), it is particularly marked in the new Fc3-4 P1

A C C E P T E D M A N U S C R I P T
14 form, and this prompted an analysis of the relative B-factors across all of the Fc3-4 and IgE-Fc structures, comparing C3 with C4 (Fig. 6A&B), and also the C4proximal and C4-distal regions of C3 (Fig. 6C&D).In order to compare B-factors from structures refined with different protocols and at different resolutions, the values for each chain or domain were normalised by dividing by the average B-factor (refer to Materials and methods).
The B-factor analysis revealed that the C3 domains showed greater intrinsic flexibility than the C4 domains in the unbound Fc3-4 and IgE-Fc structures, despite the range of different crystal packing environments (Fig. 6A).In the FcRI-bound, aFab-bound and omalizumab Fab-bound IgE-Fc structures, the opposite effect is seen, undoubtedly due to the stabilising effect of FcRI, aFab and omalizumab Fab binding to the C3 domains.In the CD23-bound, MEDI4212-bound and artificially constrained Fc3-4 crystal structures, B-factors for the C3 and C4 domains are similar (Fig. 6A).
Comparison of B-factors within the C3 domain (Fig 6B ) shows that the C4-distal region displays the greater degree of disorder or flexibility in virtually all of the structures, including IgE-Fc and also the Fc3-4/CD23 complexes.The C4 domains, through extensive interactions with C3, stabilise the C4-proximal regions of C3, but the C2 domains in IgE-Fc do not have the same effect upon the C4-distal region (Fig. 6A).In the FcRI-bound, the extended aFab-bound and the omalizumab Fabbound IgE-Fc structures, the C4-distal region of C3 is stabilised, unsurprisingly, since this is the location of the two receptor sub-sites, one in each C3, and the aFab binding site [4,5]; the omalizumab Fab epitope is located on the exposed outer face of the C3 domain [26].The pattern for MEDI4212-bound Fc3-4 is again different, but its epitope principally involves the C4-proximal region of C3.
This flexibility of the C3 domains in the context of IgE-Fc and Fc3-4 and their stabilisation by FcRI-binding is consistent with the more extreme behaviour of the C3 domain when studied in isolation: alone it behaves as a partially folded "molten

A C C E P T E D M A N U S C R I P T
globule" [7,8,9,10], yet it can bind sFcRI [11,7] and, in its presence, adopt a more folded structure [8,9].

Differential stability of the IgE-Fc domains
The thermally-induced unfolding of Fc3-4 and IgE-Fc in the presence of increasing concentrations of urea was measured by DSF (Fig. 7).The Fc3-4 data show a single unfolding event at all urea concentrations.The unfolding event occurs at 52C in the absence of urea, and at lower melting temperatures as the urea concentration is increased (Fig. 7, Table 2).This implies that the C3 and C4 domains unfold cooperatively.The IgE-Fc data however shows a two-state unfolding (Fig. 7), the first event, in the absence of urea, at 55C (T m 1) and the second at 64C (T m 2).The latter is taken to be the unfolding of the C2 domains, occurring after that of C3 and C4.
Both T m 1 and T m 2 decrease with increasing urea concentration (Fig. 7, Table 2).For Fc3-4, and also for these domains within IgE-Fc, 4M urea causes complete unfolding in the absence of heating, whereas >6M urea is required to achieve this for the C2 domains.
The intensity of the signals from Fc3-4 are consistently smaller than those from IgE-Fc (at the same concentration), which may be due to differences in the aggregation properties of the two proteins as denaturation occurs.Aggregation reduces the hydrophobic surface area to which Sypro-orange molecules can bind, thus reducing the signal.The C3 domains, and in particular the N-terminal regions, are more flexible (as seen in crystal structures) and perhaps inherently more prone to unfolding in Fc3-4 compared with IgE-Fc, thus promoting aggregation of the former.However, as seen in the B-factor analysis (Fig. 6A), the stabilising effect of the (C2) 2 domain pair upon the Fc3-4 region is relatively modest, since the T m and T m 1 values for Fc3-4 and IgE-Fc respectively are very similar under all urea conditions (Table 2).

Conclusions
The C3 domains are central to the function of IgE since they contain the receptorbinding sites for both FcRI and CD23.These two sites are located at opposite ends

A C C E P T E D M A N U S C R I P T
16 of the C3 domain, yet their binding is mutually incompatible [6,28].Allosteric communication between these two sites clearly involves quaternary structural changes in IgE-Fc and Fc3-4 domains, predominantly in the relative disposition of the C3 domains [6,28].However, the C3 domain has been identified as an outlier according to various measures of folding and stability [10], and it may be that this unique behaviour is required in order to transmit the allosteric signal.The three new Fc3-4 crystal structures reported here contribute to understanding both the intrinsic flexibility of the C3 domains and their quaternary structure, and we have now compared all of the (34) available IgE-Fc and Fc3-4 structures.This highlights the more extreme conformational variation in C3 than was seen in an earlier analysis based upon the far fewer structures then available [18].Furthermore, the enhanced resolution for two of the structures reported here provides new detail concerning the ordered regions of the N-linked carbohydrate that is attached to this intrinsically flexible domain.
Analysis of the B-factors from all the published crystal structures together with the new structures presented here confirms the enhanced intrinsic flexibility of the C3 domains and demonstrates that this is most pronounced in their C4-distal regions.
The ability to average over so many different structures in different crystal forms, overcomes any local effects of crystal packing constraints.The C3 domains are stabilised by FcRI binding (Fig. 6), consistent with the results of experiments conducted with isolated C3 domains binding to sFcRI [8,9] and with thermal stability data reported for IgE-Fc with and without sFcRI [44].
The thermal stability data that we present here implicate the C3 domains as the most susceptible to denaturation within IgE-Fc.The thermal lability of IgE has been recognised for a long time.Even before the recognition of IgE as an antibody, the loss of "reaginic" activity in atopic sera by heating to 56C was reported [29], and shortly after the discovery of the IgE antibody class, this unusual thermal susceptibility compared with IgG (for which T m values are typically ~72C), was confirmed [30].
Furthermore, on the basis of circular dichroism spectroscopy and retention or loss of antigenic determinants as a function of temperature, Dorrington and Bennich were able to write that "the irreversible changes seen with intact IgE result from structural

A C C E P T E D M A N U S C R I P T
changes in that portion of chain C-terminal to Fc''- [their terminology for the C2 domains]", i.e. in the C3-4 domains [30].We show here that IgE-Fc unfolds in a two-step process, the first involving the Fc3-4 region with T m values similar to that of intact IgE, and the second involving the C2 domains.Since the T m 1 and T m values for IgE-Fc and Fc3-4 respectively are very similar, the C2 domains have only a marginal influence on the thermal stability of the C3 domains.
In IgG-Fc, glycosylation of the complex type, N-linked at the structurally homologous Asn297 in each C2, contributes to the thermal stability of this domain, and the two domains have distinct T m values: glycosylated, 71C and 82C for C2 and C3 respectively; fully deglycosylated, 66C and 82C [45].In contrast, Fc3-4 unfolds cooperatively at the much lower temperature of 52C even when fully glycosylated (Fig. 7 and Table 2).Deglycosylation of Fc3-4 further reduces the T m by 4C [46].
The inability of the high-mannose carbohydrate to stabilise the C3 domain is consistent not only with its intrinsic lability, but also with the remarkable observation that at the point of covalent connection to Asn394 in C3, in chain C of the Fc3-4 P2 1 "large" form and chain F of the P1 form, the flexibility and/or disorder is so great that there is no visible electron density (Fig. 3B&D); this is despite the fact that, as seen most clearly in the structure reported here, well-ordered carbohydrate units make contact with C4 (Fig. 4).In IgG-Fc, no carbohydrate contact is ever made with the C3 domains.This difference between IgG and IgE is also reflected in the functional requirement for Fc glycosylation.Whereas substantially reduced glycosylation compromises IgG-Fc receptor-mediated effector functions [47], glycosylation at Asn394, while required for folding and secretion of IgE in vitro and in vivo [15], is not essential for either FcRI or CD23 binding, since refolded IgE-Fc lacking carbohydrate altogether has native-like affinity for both receptors [48,49,50,51].
The relationship between glycosylation, structure and function in IgE is thus very different to IgG, and appears to be yet another unique feature of this class of antibody.

ACCEPTED MANUSCRIPT
A

2 Man 5 .
In the Fc3-4 P1 structure: chain A GlcNAc 1 Man 3 ; chain B GlcNAc 2 Man 3 ; chain C GlcNAc 2 Man 3 ; chain D GlcNAc 2 Man 5 ; chain E GlcNAc 2 Man 3 ; chain F GlcNAc 0 Man 3 .For the quaternary structural comparisons, the angle defined by the three C atoms X337-X497-Y337 or Y337-Y497-X337 (where X and Y refer to two ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T paired chains) was measured using the PyMOL Molecular Graphics System (Version 1.8.2.1 Schrödinger, LLC).NB: where residues 337 or 497 were not modelled in the structure (Fc3-4 P1 structure chains A and B, structure 5ANM chain A, B, C and D domains.For example, in IgE-Fc chain B a terminal mannose residue, Man 952, forms a hydrogen bond with the main-chain of Arg342.Hydrogen bonds mediated by water molecules are also formed between: Man952 and Ser344, Ser341, Ile474 and Ser475; Man953 and Thr492 (Fig.4A).In the Fc3-4 P2 1 'large' structure chain D, a terminal mannose residue, Man949, forms hydrogen bonds with main-chain atoms of Arg342 and Ile474; Man950 forms hydrogen bonds with Thr492; Man947 forms hydrogen bonds with Gln494.Hydrogen bonds mediated by water molecules are also formed between: Man948 and Ser341, Thr357, Ile474 and Thr493; Man949 and Arg342, Asp347, Asp473, and Ser475; Man950 and Thr492 (Fig.4B).With theACCEPTED MANUSCRIPTA C C E P T E D M A N U S C R I P Texception of Man947-Gln494, these hydrogen bonds are not seen in the other chains, or in any of the previously published Fc3-4 or IgE-Fc structures.
complexes with FcRI, and IgE-Fc bound to the omalizumab Fab, are the most open, ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T 13 while those bound to CD23 and the numerous uncomplexed Fc3-4 structures show how they almost span the entire range from closed to the open conformation in the FcRI complex: none however is as open as that seen in the omalizumab Fab / IgE-Fc complex.In contrast to Fc3-4, there is to date only a single crystal form of uncomplexed IgE-Fc despite extensive crystallisation trials (authors' unpublished observations).This may imply that the C2 domains stabilise, through extensive contact with one of the C3 domains, an intermediate quaternary structure (between open and closed) [17].The recent structure of the complex between IgE-Fc and two anti-IgE aFab molecules unexpectedly revealed that the C2 domains can also adopt an extended, linear conformation with respect to the Fc3-4 region, with no C2/C3

Fig. 2 .
Fig. 2. Carbohydrate structures in the four crystal forms.(A) Mass spectrometry trace showing carbohydrate present in IgE-Fc.The molecular mass of the polypeptide dimer is 72 604 Da, and with two N-acetylglucosamine (GlcNac) residues per chain is 73 416 Da.The first peak in the spectrum corresponds to one additional mannose (Man) residue in each chain.(B) Mass spectrometry trace showing carbohydrate present in Fc3-4.The molecular mass of the polypeptide dimer is 49 756 Da, and with two GlcNac residues per chain is 50 568 Da. (C) Blue squares represent GlcNAc and green circles, Man units.i) Shows the arrangement of the high-mannose N-linked carbohydrate modelled in IgE-Fc chain B, Fc3-4 P2 1 'large' chains B and D, and Fc3-4 P1 chain D. ii) Shows the carbohydrate modelled in Fc3-4 P2 1 'small' chains A and B, and in Fc3-4 P1 chains C and E. iii) Shows the carbohydrate modelled in IgE-Fc chain A and Fc3-4 P1 chain B. iv) Shows the carbohydrate modelled in Fc3-4 P2 1 'large' chain A. v) Shows the carbohydrate modelled in Fc3-4 P2 1 'large' chain C and Fc3-4 P1 chain A. vi) Shows the carbohydrate modelled in Fc3-4 P1 chain F.

Fig. 3 .Fc3- 4
Fig. 3. Electron density for carbohydrate at Asn394 in chains E and F of the Fc3-4 P1 crystal form, and chains C and D of the Fc3-4 P2 1 'large' crystal form.(A) Chain E of the Fc3-4 P1 form: the covalent connection between Asn394 and GlcNAc has well defined density.(B) Chain F of the Fc3-4 P1 form: Asn394 has poorly defined density and the two GlcNAc units are missing.(C) Chain C of the

Fig. 4 .
Fig. 4. Contacts between carbohydrate and the C3 and C4 domains in the IgE-Fc crystal structure chain B and the Fc3-4 P2 1 'large' crystal form chain D. (A) IgE-Fc crystal structure chain B showing interactions between: Ser341, a water molecule and Man952; Arg342 and Man952; Ser 344, a water molecule and Man952; Ser475, a water molecule and Man952; Ile474, a water molecule and Man952; a polyethylene glycol molecule (PEG4), a water molecule and Man953; Thr492, a water molecule and Man953.(B) Fc3-4 P2 1 'large' crystal form chain D showing interactions between: Thr357, three water molecules and Man948; Ser341, a water molecule and Man948; Arg342, a water molecule and Man949; Arg342 and Man949; Asp347, two water molecules and Man949; Asp473 a water molecule and Man949; Ser475 a water molecule and Man949; Ile474 and Man949; Ile474, three water molecules and Man948; Thr492 and Man950; Thr492, a water molecule and Man950; Thr493, two water molecules and Man948; Gln494 and Man947.Hydrogen bonds are shown as grey dotted lines and the black crosses are ordered, bridging water molecules.Carbohydrate residues (shown as sticks) are coloured by atom type, and the C3 and C4 domains are coloured pink and blue, respectively.

6
Here we report two new crystal forms of Fc3-4 (at 2.26Å and 2.2Å resolution) together with one of the earlier forms now solved at the highest resolution yet reported (2.0Å) and IgE-Fc also solved at the highest resolution yet reported (1.75Å);

Table 1 .
Data processing and refinement statistics Values in parentheses are for the highest resolution shell b R free set comprises 5% of reflections c ethylene glycol, polyethylene glycol, phosphate a d ethylene glycol, polyethylene glycol e ethylene glycol, polyethylene glycol, sulphate f ethylene glycol, polyethylene glycol