The IgE residue numbers employed follow the KABAT numbering24, which is not contiguous. To distinguish omalizumab residues from IgE ones, the former will be preceded by the complementarity-determining region (CDR) loop; e.g., L3:His92 indicates His92 from the CDR-L3 loop.

The crystal structure of omalizumab Fab

The 2.42-Å crystal structure of the omalizumab-Fab region shows a highly negatively charged surface (see Fig. 3a, which shows the surface electrostatic potentials from the APBS program25 for the six CDRs). The L1, L2 and L3 CDRs exhibit negative surface potentials, whereas the heavy chain CDR loops are neutral. Surprisingly, the three histidines in the H3 loop (His97, His100a and His100c), which were assumed to be positively charged in previous works14,26, are predicted to be neutral by five programs (Reduce27, Whatif28, PDB2PR29, PROPKA330 and HAAD31). The neutral state of H3:His100c is consistent with its low solvent-accessible surface area (SASA) of 3% in the crystal structure. Although H3:His100a (SASA = 15%) and H3:His97 (SASA = 31%) are partially solvent exposed, they are hydrogen-bonded to each other and well-packed with vdW contacts to nearby hydrophobic residues including H3:His100c (Fig. 3b). The H3, L1 and L3 loops contain residues implicated in direct/indirect binding to IgE from site-directed mutagenesis studies26, namely, H3:His97, H3:His100c, L1:Asp30, L3:Glu93 and L3:Asp94.

Figure 3 The omalizumab-Fv region. (a) Electrostatic potentials derived from the 2.42 Å crystal structure; arrows indicate the residues implicated in binding IgE from site-directed mutagenesis studies. (b) Packing and hydrogen-bonding interactions of the three histidines in the H3 loop. (c) Interactions of L1:Asp30 showing that its side chain points away from the protein surface. Omalizumab-Fv residues are in green and IgE residues in blue. Full size image

The docked IgE-Fc/omalizumab-Fab structure is consistent with available experimental data

To obtain a structure of omalizumab bound to IgE, the crystal structures of omalizumab-Fab and human IgE-Fc (PDB 4gt7 and 4j4p) consisting of the Cε2, Cε3 and Cε4 domains (denoted as Cε2-3-4) were docked together, as described in Methods. The predicted IgE-Fc/omalizumab-Fab structure is consistent with the following experimental data:

1 The docked IgE-Fc/omalizumab-Fab structure shows that L3:Glu93, whose mutation along with L3:Asp94 to Ala reduced binding to IgE26, forms a salt bridge with Arg457 in the Cε3 domain. 2 It also shows that all the IgE residues experimentally implicated in binding omalizumab are within 5 Å of the omalizumab residues. These IgE residues are Ser407, Arg408, Ser411, Lys415, Glu452,455QCRVT459, Arg465 and Met469 whose mutation to Glu407/Gln407, Glu408, Gln411, Asp415, Arg452/Gln452, 455ACAVA459, Glu465 and Ala469 significantly reduced or nearly abolished binding to omalizumab14. They also include the 462HLP464 motif determined from fine epitope mapping of omalizumab32. 3 The orientation of omalizumab-Fab bound to IgE allows for two omalizumabs to bind to a single IgE (Fig. 4a), in agreement with experimental results33. Figure 4 The IgE/omalizumab interface. (a) Two omalizumab-Fab molecules (green) binding to two IgE Cε3-4 domains (marine/blue) with the Cε2 domains (cyan) in the extended conformation. (b) Omalizumab-binding site (yellow/wheat) on the IgE Cε3 domain. Full size image 4 The omalizumab-binding site on IgE is near the binding sites for CD23 and FcεRI. 5 The total SASA change upon binding the omalizumab-Fab and IgE-Fc (2,054 Å2) is consistent with the buried surface areas (1,144–2,500 Å2) computed for 22 Ab/Ag complexes in the protein-protein docking benchmark version 3.034.

The IgE/omalizumab interface

The IgE-Fc/omalizumab-Fab structure consistent with available experimental data was used as the starting point for four sets of MD simulations in explicit water to create an ensemble of conformations, as described in Methods. These conformations were used to compute average distances between IgE and omalizumab heavy atoms. An interface residue is defined by a mean heavy–heavy atom distance ≤5 Å between IgE and omalizumab. A hydrogen bond is defined by a mean hydrogen–acceptor atom distance ≤2.4 Å and a donor–hydrogen–acceptor angle >130o, while a vdW contact is defined by a mean heavy–heavy atom distance ≤4.0 Å at least 50% of the time in two or more simulations.

The omalizumab interface residues in the IgE/omalizumab complex are distributed among all the CDRs except the L2 loop (Supplementary Table S1). Missing from the IgE/omalizumab interface are H3:His100c and L1:Asp30, which have been implicated in binding IgE from mutagenesis studies26 (see above). These two omalizumab residues appear to play a conformational role in binding to IgE: In the MD structures, the buried H3:His100c forms vdW contacts with L:Tyr49, H3:Ser96 and H3:Phe99 and a backbone-backbone hydrogen bond to H3:His100a, which in turn is hydrogen-bonded to H3:His97 and H3:Ser96 (Fig. 3b). This well-packed core helps to rationalize why simultaneous mutation of the three His residues in the H3 loop to Ala abolished binding to IgE26. The L1:Asp30 side-chain points towards the protein interior in the X-ray structure and is hydrogen-bonded to L1:Ser31, while its backbone is in vdW contact with L1:Tyr27D and L1:Asp28, which binds IgE via hydrogen bonds with Ser471 and Arg470, respectively. Thus, L1:Asp30 plays a role in stabilizing the L1 loop for binding omalizumab, hence, its mutation to Ala would destabilize the L1 loop, accounting for the observed loss of binding to IgE26.

The IgE interface residues in the IgE/omalizumab complex stem from two nearly linear epitopes (Supplementary Table 1): The first nearly contiguous sequence involves 405T-SR408 from β-strand C and 410ASGKP416 in the following CD loop (there are no residues with KABAT numbers 409, 412 and 413; a dash indicates absence of the residue at the interface). This epitope forms hydrogen bonds or vdW contacts with the L3, H2 and H3 loops. The second nearly contiguous sequence consists of Glu450 from helix-B, 451GET453 in the EF' turn, 455Q-R-T459 in β-strand F, 460HPHLPRA466 in the FG loop and 467LMRS471 in β-strand G (there is no residue with KABAT number 468). This epitope forms hydrogen bonds or vdW contacts with the L1, L3 and H2 loops. Although the interface residues are formed from two disparate sequences, they are located together with β-strands C, F and G forming an anti-parallel β-barrel structure (Fig. 4b). Notably, the interface residues encompass all residues experimentally implicated in binding omalizumab14,32 (see above).

Roles of key IgE residues in binding omalizumab

Which residues make the most favorable contributions towards binding omalizumab? To address this question, the binding free energy contribution of each IgE residue was computed using 8,000 conformations sampled from four simulations of the Cε3-4 dimer bound to omalizumab-Fv in explicit water. Although the scheme used to compute the binding free energy cannot yield accurate absolute free energies due to the continuum solvent approximation used to compute the interaction free energy35 (see Methods), it can yield trends in the relative free energy contributions of residues towards binding a given ligand. The free energy contributions of non-interface residues are insignificant, so only those of the interface residues are listed in Supplementary Table 1. Among the IgE interface residues, Ser407, Ala410, Ser411, Lys415, Arg457, Arg465, Met469 and Arg470 make significantly favorable contributions to binding omalizumab (see Fig. 5a). These residues have been implicated in binding omalizumab from site-directed mutagenesis26 except Ala410 and Arg470 whose roles in binding omalizumab have not been experimentally examined. The 462HLP464 motif is also experimentally implicated in binding omalizumab, but its net free energy contribution is relatively small (–2.3 kcal/mol).

Figure 5 Free energy contributions of IgE residues towards binding (a) omalizumab-Fv, (b) CD23 and (c) FcεRI. Residues experimentally implicated in binding omalizumab are labeled. Full size image

Although mutagenesis studies reveal that Ser407, Arg408, Ser411, Lys415, Glu452 455QCRVT459, Arg465 and Met469 reduced or nearly abolished omalizumab binding (see above), they cannot discern if these residues directly contact omalizumab or provide some conformational stabilization. The roles of these residues can be deduced from their interactions and free energy contributions towards binding omalizumab: Ser407, Lys415, Arg457, Arg465 and Met469 directly hydrogen bond to omalizumab and make significant binding free energy contributions. On the other hand, Arg408 and Glu452 stabilize the IgE conformation critical for binding, but do not directly contact omalizumab and make negligible (–1 kcal/mol) binding free energy contributions. These two residues are salt-bridged to each other and indirectly bind omalizumab: Arg408 forms a side chain–backbone hydrogen bond with Lys415, which in turn is salt-bridged to H2:Asp54, while Glu452 forms a side chain–side chain hydrogen bond with Ser411, which is in vdW contact with H2:Asn58.

The interactions found are consistent with and help to rationalize the mutagenesis results. For example, the Ser411---Glu452---Arg408---Lys415---H2:Asp54 hydrogen-bonding network found in the MD simulations can explain why mutation of Arg408 to Glu or Glu452 to Arg significantly reduced binding to omalizumab14: These mutations result in repulsive Glu408---Glu452 and Arg452---Arg408 interactions, resulting in conformational changes that hinder IgE from binding omalizumab. Likewise, mutation of Ser407, Lys415 and Arg465 to an acidic residue (Glu or Asp) nearly abolished binding to omalizumab14, as these three residues interacted with omalizumab Asp residues during the simulations: Ser407 formed transient hydrogen bonds with L3:Asp94, Lys415 is salt-bridged to H2:Asp54, while Arg465 is salt-bridged to L1:Asp27C. The loss of binding to omalizumab upon mutation of 455QCRVT459 to 455ACAVA45914 can be attributed mainly to Arg457, which formed side chain–side chain hydrogen bonds with the L3:His92 and L3:Glu93 in the simulations. On the other hand, the loss of binding to omalizumab upon mutation of Met469 to Ala14 is likely due to loss of packing interactions, as the Met469 side chain formed multiple vdW contacts with L1:Tyr27D, L1:Tyr32 and L3:His92 in the simulations. In contrast to Met469, mutation of the neighboring Ser471 to Ala did not significantly affect omalizumab binding14, in line with its insignificant free energy contribution (–0.8 ± 0.8 kcal/mol), despite its direct omalizumab contact via a sidechain–sidechain hydrogen bond with L1:Tyr27D.

Key IgE residues involved in binding the IgE receptors

To determine if the IgE residues involved in binding omalizumab are also crucial for binding CD23 or FcεRI, the free energy contributions from the Cε3-4 residues towards binding either IgE receptor were computed from four independent simulations of the Cε3-4 dimer bound to CD23 or FcεRI (see Methods). The MD simulations could maintain the structural integrity of the IgE/receptor crystal structures: The Whatif program28 indicated fourteen potential hydrogen bonds between the IgE and its receptor in the X-ray structure of IgE in complex with CD23 (PDB 4gko)18 and nine for FcεRI (PDB 1f6a)15. In each case, all but two putative IgE---receptor hydrogen bonds in the crystal structures were preserved in at least two simulations (Supplementary Table S2). The free energy contributions from the IgE interface residues upon binding to CD23 (Fig. 5b) and FcεRI (Fig. 5c) reveal the most important IgE regions involved in binding the IgE receptors and those residues that are shared by the low/high-affinity receptor and omalizumab.

CD23

The IgE/CD23 interface residues are found mainly in four sequential regions encompassing the Cε3-4 domains from one of the heavy chains (Supplementary Table S3a). Two regions consisting of 408RASxK415 and 446RDxIEGE452 contains crucial omalizumab-binding residues (bold), notably Lys415 and Glu452, whose mutations to Asp and Arg, respectively, nearly abolished binding to omalizumab14. Lys415 is salt-bridged to CD23:Asp193, while Glu452 is hydrogen-bonded to neutral CD23:His186. Apart from binding CD23, Glu452 also links the two regions containing omalizumab-binding residues via hydrogen bonds with Ser411. These two regions make significantly favorable free energy contributions (–8 and –13 kcal/mol) to binding CD23.

The other CD23-binding regions do not involve omalizumab-binding residues. One of them consists of Lys474 and Ser476 in the Cε3–Cε4 linker followed by 497GPRAA501 in the Cε4 domain. This region (in particular Lys474, Ser476 and Arg499) makes a large free energy contribution to binding CD23 (–25 kcal/mol) that is comparable to the CD23-binding regions encompassing omalizumab-binding residues (–21 kcal/mol). In contrast, the other “unique” CD23-binding region, which consists of 592EAASxSQ598 in the Cε4 domain, makes a much smaller binding free energy contribution (–4 kcal/mol). These two CD23-specific regions are linked by salt bridges between Glu592 and Lys474/Arg499.

FcεRI

Unlike CD23, which binds to residues in the Cε3 and Cε4 domains belonging to the same IgE chain, the FcεRIα domain binds to residues in both Cε3 domains but not to residues in either Cε4 domain (Supplementary Table S3b). The FcεRIα-binding residues are found in four sequential regions, two of which are partially duplicated on the second chain. The two duplicated FcεRIα-binding regions contain residues found at the IgE/omalizumab interface. One of these “duplicated” regions is 460H-HL463 in chain B and the 461PHLPR465 motif in chain A. The latter makes a large favorable free energy contribution (–17 kcal/mol) towards binding FcεRIα with Pro464 contributing slightly over half (–9 kcal/mol). The 462HLPR465 motif has been experimentally implicated in binding omalizumab (see above). Notably, His462, Pro464 and Arg465 form hydrogen bonds with FcεRIα Trp110, Ser85 and Asp86 side chains, respectively. Interestingly, omalizumab seems to mimic the interactions made by FcεRIα with Pro464 and Arg465, as L1:Ser27A and L1:Asp27C hydrogen bond to Pro464 and Arg465, respectively. The other “duplicated” region is the 365RGV367 motif in chain B, which is contained in the longer 363NPRGVA368 motif in chain A. These two motifs make significant favorable free energy contributions (–5 and –11 kcal/mol) towards binding FcεRIα with Arg365 making the largest contribution. Pro364 from chain A and Arg365 from chain B are close to omalizumab. The other two FcεRIα-binding regions comprising 394DLAPS398 and 428RNGT434 in IgE chain B, which do not involve omalizumab-binding residues, also make quite favorable free energy contributions (–12 and –7 kcal/mol) towards binding FcεRIα.