mAb806 binding to epidermal growth factor receptor: a computational study.

Research Article

Proteins: Structure, Function and Bioinformatics DOI 10.1002/prot.24714

mAb806 binding to EGFR: a computational study

Yao Zong Ng1#, Srinivasaraghavan Kannan1, David P Lane2, Gloria Fuentes1¥*, Chandra Verma1, 3, 4*

1

Bioinformatics Institute (A*STAR), 30 Biopolis street, #07-01, Matrix, Singapore 138671

2

p53 Laboratory (A*STAR), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, Singapore 138648 3

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543

4

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 *Corresponding authors: Gloria Fuentes, Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore, E-mail: [email protected]. Chandra S. Verma, Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore, E-mail: [email protected]; Tel: +65 6478 8273; Fax: +65 6478 9048. # Current address: Department of Chemistry, Columbia University, New York, NY 10027, USA. ¥ Current address: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan.

Keywords: egfr, extracellular domain, mab806, epitope, modelling, md simulations Conflict of Interest: None Financial Disclosures: Financial supported from Biomedical Research Council (BMRC), Agency for Science, Technology and Research (A* STAR), Singapore, is gratefully acknowledged. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/prot.24714 © 2014 Wiley Periodicals, Inc. Received: Jun 19, 2014; Revised: Oct 13, 2014; Accepted: Oct 24, 2014

PROTEINS: Structure, Function, and Bioinformatics

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Abstract The epidermal growth factor receptor (EGFR) is an important target in the treatment of cancer. A very potent antibody, mAb806, has been developed against overexpressed EGFR and was found to be particularly active in brain tumours. Structural studies reveal that it binds an epitope on the extracellular region of the EGFR. However, this epitope is cryptic / buried in crystal structures of the active (untethered) and inactive (tethered) EGFR and it is unclear as to how the antibody interacts with this region. To explore this interaction we combined molecular docking, steered molecular dynamics and equilibrium molecular dynamics simulations. Our computational models reveal that the antibody induces local unfolding around the epitope to form the antibody – EGFR complex. In addition, regions in the vicinity of the epitope also modulate the interaction, which are in accord with several other known antibody-antigen interactions, and offers new possibilities for the design of antibodies with increased potency and specificity for this receptor.

2 John Wiley & Sons, Inc.

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Introduction Epidermal growth factor receptor (EGFR), a member of the Her (ErbB) receptor tyrosine kinase family, plays crucial roles in cellular pathways regulating cell proliferation, migration and differentiation.1 Deregulation of EGFR has been implicated in several diseases including glioblastoma, breast, skin and lung cancer.2 EGFR is a transmembrane protein made up of an extracellular region comprising four domains (I, II, III, IV), a transmembrane single-pass helix, a juxtamembrane region and an intracellular kinase.3 Several different ligands, sharing a common epidermal growth factor (EGF)-like fold, are known to bind to the extracellular domain of EGFR. Upon binding to the extracellular domain of EGFR, these ligands promote dimerization, leading to activation and autophosphorylation of the cytoplasmic (intracellular) kinase domains, which subsequently offer docking sites to recruit other downstream proteins. Several biochemical and structural studies have demonstrated the ligand-dependent activation mechanism of EGFR in detail.4-7 Available crystal structures have demonstrated that the extracellular region can adopt at least two conformations – an “untethered or extended” conformation in the EGF bound, active state, and a “tethered or compact” inactive conformation in the absence of ligand (Figure S1). The binding of ligands promotes large scale conformational changes, leading to EGFR dimerization.4-7 Kuriyan et al.8 have, through elegant experiments, revealed the mechanism underlying activation which involves dimerization of the cytoplasmic domains, facilitating autophosphorylation. Due to the aberrant activation of this receptor in several cancers, its inhibition has been successfully exploited for cancer treatment.9-11 Current strategies include targeting the extracellular domains with monoclonal antibodies (mAb)12-14 or the intracellular kinase domain with small molecule kinase inhibitors.15-18 Detailed characterization of the mode of interactions between the inhibitors and their targets has guided 3 John Wiley & Sons, Inc.


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the design and optimization of therapies for cancer treatment. However, most patients suffer from side effects and eventual resistance to these therapies.

Earlier, a monoclonal antibody (mAb806) was raised against a truncated form of the EGFR (∆27EGFR), which is present only in certain cancers.13 It was further observed that mAb806 also binds to over-expressed wild-type EGFR (wtEGFR) on cancer cells but surprisingly, not wtEGFR on normal cells.14 This differential binding of the antibody represents a promising development towards the alleviation of side effects that normally characterize EGFR antibody based cancer therapy. It was hypothesized that differential binding is due to altered EGFR glycosylation13 or an indirect effect of glycosylation shifting the EGFR population to states that are favourable for mAb806 binding.19 Epitope mapping studies show that mAb806 recognizes a disulphide-bonded loop in EGFR (residues 287-302; all residue numbering is based on the mature EGFR)20 and the structure of mAb806 bound to this peptide fragment has been solved by X-ray crystallography (Figure 1, PDB ig 3GV5).21 The interactions between the antibody and the epitope are made up of hydrogen bonds and a salt bridge complemented by hydrophobic packing interactions. Interestingly the conformation of the peptide fragment in the apo state of EGFR (both the tethered and untethered states of the full length extracellular domain as revealed by X-ray crystallography) and when bound to the antibody are very similar, with root mean square difference (RMSD) of < 1Å. However while this epitope is constitutively exposed in the (∆2-7EGFR) mutant, it is cryptic or inaccessible to the antibody in the full-length extracellular domain of EGFR, in both the tethered and untethered states. Superposition of the antibody (mAb806)–peptide structure with the full-length extracellular domain of EGFR in both the tethered and untethered states results in steric clashes between the antibody and the EGFR. This


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suggests that conformational changes must occur in the extracellular domain of EGFR for binding of the antibody to this epitope; there is some evidence that the antibody preferentially recognizes an untethered form of the receptor.20 However, the nature of these changes remains elusive. An appealing hypothesis is that the epitope becomes exposed partially or completely during receptor activation (the transition from tethered to untethered state), mutation or overexpression, which allows for mAb806 binding.20 This is strengthened by the observation that removal of a disulphide bond (C271-C283) proximal to the epitope by mutation of the cysteines to alanine increases mAb806 binding.21

A model of the mAb806 – EGFR complex19 was reported earlier which used a homology model of mAb806 with the peptide epitope docked; this peptide was extracted from the crystal structure of the extracellular domain of EGFR in an active conformation complexed with the EGF ligand (PDB id 1NQL). However, the epitope in this modelled mAb806 – EGFR complex adopted a ‘flipped out’ conformation, in contrast to the recent crystal structure of mAb806 with the peptide,20 which shows the epitope to be in a ‘flipped in’ conformation (Figure S2). Hence the mode of binding of the antibody to the extracellular domain of EGFR at the epitope region remains unclear.

To address this, we now combine several computational approaches to understand how the antibody binds to the epitope in the context of the full-length extracellular region of EGFR. Since the epitope region is completely buried in the available crystal structures of the full-length extracellular domain of EGFR, large conformational changes are required for binding of the antibody to EGFR. To induce exposure of this epitope to antibody binding/interactions we have



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complemented docking and molecular dynamics (MD) simulations with steered MD (SMD) simulations to guide the EGFR-antibody complex such that the interactions with the epitope are consistent with the binding mode observed in the crystal structure of the antibody–epitope complex.

Materials and Methods Structure preparation The crystal structures of the extracellular domain of EGFR in its untethered (PDB id 1IVO, resolution 3.30Å)22 and tethered (PDB id 1YY9, resolution 2.61Å)23 states and the Fab arm of the antibody mAb806 (PDB id 3G5V, resolution 2.00Å)21 were used for the docking calculations. The coordinates for the missing residues were modelled using Modeller.24 Extracellular domain IV was missing in the crystal structure of EGFR in the untethered conformation and was modelled using the tethered structure, conserving the relative orientation of domains III and IV.3 The EGFR structures contained residues 1 to 612, while the antibody contained residues 1-212 (light chain) and 301-513 (heavy chain) (renumbered by adding 300). Prior to docking, the structures were minimized using 500 steps of Steepest Descent (SD) followed by 1000 steps of Adopted Basis Newton-Raphson method (ABNR), using CHARMM (c34b2).25 The solvent accessible surface area of the epitope is ~1578 and 1637 Å2 respectively in the tethered and untethered states (it is only 1450 Å2 in a structure of EGFR complexed with adnectin that was published after the current work was performed, pdb id 3QWQ).26


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Antibody Docking Docking of mAb806 with the extracellular domain of EGFR (both tethered and untethered) was carried out using HADDOCK (High Ambiguity Driven biomolecular DOCKing).27,28 Each HADDOCK run consisted of three stages: randomization of orientations and rigid-body docking, semi-rigid simulated annealing in torsion angle space (backbone fixed, only side chains allowed to move), and final refinement in explicit solvent. A total of 1000 rigid body solutions were generated in stage one. The top 200 orientations with the lowest energy were used for the second and third stages. A set of distance restraints called Ambiguous Interaction Restraints (AIRs) between residues that are shown to be involved in the interactions were used during docking to drive complex formation. Distances between residues from the complementarity-determining regions (CDRs) of mAb806 (light chain residues 24-34, 49-56, 89-97; heavy chain residues 331336, 351-366, 397-405) and EGFR epitope residues (E293, G298, V299 and C302) that have been identified by yeast display, were used as restraints.21

The models generated by HADDOCK were clustered and the HADDOCK score for each model was calculated from a weighted sum of terms: Electrostatic energy (weight 0.2) + Van der Waals energy (weight 1.0) + Desolvation energy (weight 1.0) + Restraint violation energy (weight 0.1).

Molecular dynamics simulations MD simulations were carried out with NAMD29 using the CHARMM22 force-field.30 Simulation systems were solvated in a box of TIP3P31 water molecules with a minimum spacing of 10 Å from the box edges to the protein. The solvated systems were neutralized by adding sufficient numbers of counter ions, followed by random placement of sodium and chloride ions to produce



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a final salt concentration of 0.15 mol dm-3. Energy minimization was performed on each system in a two-step process. First the protein atoms were restrained and the water molecules were allowed to relax over 1000 steps of conjugate gradient (CG) minimization. Then the entire system was subjected to energy minimization using CG for 1000 steps. This was followed by MD simulations, for which the protein was initially harmonically restrained (25 kcal mol -1 Ǻ2) to the energy minimized coordinates, and the system was heated up to 300 K in steps of 100 K followed by gradual removal of the positional restraints and a 0.5ns unrestrained equilibration at 300K. Production runs were carried out at constant pressure and temperature (NPT ensemble) using Langevin dynamics.

The Particle Mesh Ewald (PME)

32

method was employed to treat long-range electrostatics.

Short-range electrostatics and van der Waals forces were switched at 10 Å with a non-bonded interaction cutoff of 12 Å and scaled 1-4 non-bonded interactions were excluded. The SHAKE algorithm33 was used to restrain all hydrogen atoms enabling an integration time step of 2.0fs. Harmonic restraints with a force constant of 10kcal/mol were imposed on the backbone atoms (N, CA, C, O) of the extracellular domain IV of EGFR (residue 481-612) to mimic attachment to the lipid membrane. This helped to reduce the fluctuation of domain IV, particularly for the untethered EGFR.

Steered molecular dynamics Equilibrated structures of docked models of the EGFR–antibody complexes were subjected to SMD simulations. During SMD, a set of distance restraints between antibody and peptide epitope atoms that were derived from the crystal structure of the antibody–peptide complex were


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imposed using the collective variables (colvar) module in NAMD.29 For each model, SMD simulations were carried out with two sets of distance restraints, Set 1 and Set 2 (Table S1). The distances between the above pairs of atoms were determined for the crystal structure (PDB id 3G5V) and the untethered and tethered EGFR models after equilibration. These were set as the final distance restraints (target centers) and the initial distances respectively. The scaled force constant was set at 100kcal/mol and the number of steps for steering was set at 5000000 (10 ns). The actual force constant for each pair of atoms can be calculated by taking the scaled force constant divided by the inter-atomic distance squared. Positional restraints were imposed on the backbone atoms of extracellular domain IV of EGFR (residue 481-612) to mimic attachment to the lipid membrane.

Binding energy calculations The binding energy/interaction energy between the EGFR and antibody was calculated using the ENERGY module in CHARMM.34 The enthalpic energy was calculated as the sum of van der Waals energy, electrostatic energy, desolvation energy and internal energy, determined using an implicit Generalized Born solvation model with a simple SWitching function (GBSW)35 implemented in CHARMM. Binding energies were calculated by subtracting the sum of the energies determined separately for the antibody and EGFR, from the energy for the antibodyEGFR complex.

Hydrogen bond and hydrophobic contact analysis Hydrogen bond (D-H…A) analysis was performed using the HBOND coordinate manipulation command (corman) 34 in CHARMM. We used a distance cut off of 2.4 Å without any angle



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cutoff between the donor (D) and acceptor (A) atoms. The average number of hydrogen bonds per residue was calculated from the sum of hydrogen bonds for that residue over all frames divided by the number of frames used. The water-mediated hydrogen bonds (D-H…H2O…H-D) were calculated using the BRIDGE option. The distance cutoff between the two donor hydrogens was set at 3.9 Å, assuming tetrahedral geometry about the O from water and an O…H distance of 2.4 Å. The hydrophobic contacts were calculated using the COOR CONTACT command and defined as two carbons having a maximum distance of 3.4 Å. Inter-atomic distances were calculated using the COOR AXIS command in CHARMM to generate the inter-atomic vector and RAXI to get the vector length. The distances between C-alpha atoms of EGFR were calculated using the COOR DIST command in CHARMM. The heatmap was generated using the program R.36 Simulation trajectories were visualized using VMD37 and the figures were generated using Pymol.38

Results Conformational dynamics of the extracellular domain of EGFR In the absence of ligands, the extracellular domain of EGFR adopts a “tethered or compact” conformation (Figure S1), in which domains I and III are separated and domain II is in close association with domain IV; the dimerization arm of domain II interacts with domain IV. The tethered conformation of EGFR prevents dimerization and remains inactive. Binding of the ligand between domains I and III, promotes large scale conformational changes and the EGFR adopts an extended conformation (Figure S1) in which domain II moves away from domain IV and the exposure of the dimerization arm in domain II enables EGFR dimerization.

This

activation mechanism has been studied in great detail.4,5 Rmsd of the MD-sampled 10 John Wiley & Sons, Inc.

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conformations (Figure S3) of EGFR in both the tethered and untethered states shows that extracellular domains are conformationally highly flexible.

Simulation initiated from the

extended conformation demonstrated a significant conformational change in which the Cterminus of the extracellular domain showed large deviations (up to ~8Å, Figure S3) with respect to its starting structure.

In both simulations, domain I remains stable (structural deviations less than ~4Å, Figure S4). In the simulations initiated from the tethered state, domains II and IV remain in contact, occluding the dimerization arm (structural deviations up to ~ 6Å, Figure S4). In contrast, in the simulations initiated from the extended state, domains II and IV exhibit structural deviations in the range of ~ 10-12Å (Figure S4), thus suggesting that the extended conformation of the extracellular domain of EGFR in the absence of ligand is highly dynamic. Recently Arkhipov et al.39 have shown, through microsecond MD simulations, that the extended or untethered conformation of EGFR adopts a collapsed state, supporting the hypothesis that extracellular domains in monomers largely adopt compact conformations. Overall, domain IV exhibits large fluctuations in both the simulations. Since domain IV in EGFR is directly linked to the transmembrane domain, this domain is not expected to undergo large conformational changes upon activation by ligand binding.

To mimic this, simulations were performed by restraining domain IV to its

experimental conformation; the flexibility of domain IV was now attenuated in both states (Figure 2, Figure S4). Despite the high flexibility in both states, no conformational switch between the extended and the tethered states was observed during the simulations. MD simulations of the crystal structure of the antibody-epitope peptide showed that in their



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complexed states, both antibody and peptide were stable with rmsd from the crystal structure remaining within < 4Å (Figure 3).

Antibody docking to the extracellular domain of EGFR The experimental structure of the antibody bound to the extracellular domain of EGFR is not available, and was constructed by docking them together using the program HADDOCK (High Ambiguity Driven biomolecular Docking). For docking, the crystal structures of mAb806 (PDB id 3G5V) and of the extracellular domain of EGFR in the tethered (PDB id 1YY9) and untethered (PDB id 1IVO) conformations were used. In accordance with the available experimental structure and mutagenesis data (Figure 1), restraints were imposed between the EGFR epitope region and the complementarity-determining regions (CDRs) of the antibody to drive complex formation during docking. It is clear that for a large antibody, the interactions between the complementarity-determining regions (CDRs) of the antibody and the largely inaccessible epitope (Figure S1) would require substantial conformational rearrangements. The docking protocol (using the restraints mentioned above) was tested first by docking the peptide epitope to the antibody. This successfully reproduced the experimental bound conformation of the epitope (PDB id 3G5V) with an rmsd of ~1Å from the crystal structure as the top scored solution. Subsequent docking runs were performed for the tethered and untethered conformations. Docking was carried out in three stages as mentioned in Methods. Clustering based on RMSD of the final docked models yielded 5 major clusters of conformations (Figure 4). The clusters of docked models of untethered EGFR–antibody complex have a better HADDOCK score and larger buried surface area (data not shown) than the tethered EGFR–antibody complex. Our docking results suggest favorable association between antibody and EGFR in its untethered


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conformation which is in agreement with earlier experimental20 and computational studies19. However the conformation of the epitope in the docked models of full length extracellular domain of EGFR with antibody was quite different from the conformation of the epitope observed in the crystal structure of the antibody–epitope complex (Figure 5). The rmsd of the epitope in the initial docked models of the antibody–EGFR complexes had an rmsd of 15.3Å (untethered EGFR) and 17.1Å (tethered EGFR) relative to the crystal structure (PDB 3G5V) of the antibody–epitope complex; this values appears to be large because the superposition was carried out using only the antibody coordinates of the docked and the crystal structures (as we will see later, the epitope remains similar to that seen in the crystal structure). To evaluate the stability and to further refine the docked models of both tethered and untethered EGFR–antibody complexes, the best model, i.e. the one with the lowest HADDOCK score from the most populated cluster, was subjected to MD simulations. During the MD simulations, both the models stayed relatively stable and close to their starting conformations. The bound conformation of the antibody (Figure 6) also remained stable throughout the simulations. However the epitope region showed increased flexibility in both these docked models (Figure 6), resulting from conformational adaptations of the epitope and surrounding regions to accommodate the antibody. However the crystallographic bound conformation of the epitope was never sampled during the unrestrained simulations of the docked antibody–EGFR models. The rmsd of the epitope sampled during the simulations of the antibody–EGFR in both the tethered and untethered states stays at ~15Å (Figure 6) with respect to the crystal structure.

Steered molecular dynamics



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The above docking experiments failed to bring the antibody into close proximity to the buried epitope, as seen in the crystal structure. Neither did the subsequent MD simulations of the docked EGFR–antibody models reveal any major conformational changes, and the epitope remained inaccessible to the antibody during the simulations (Figure 6) suggesting that perhaps much longer simulations may be needed. To overcome this, we next guided the process of complex formation towards the binding mode observed in the crystal structure, through steered MD simulations (SMD). The steering is carried out by guiding the system such that during complexation, the interactions between the antibody and the epitope region observed crystallographically are reproduced. This process is carried out by restraining the distances between the sets of residues in the antibody and in the epitope that interact as observed crystallographically. For each docked model obtained above, SMD simulations were carried out with two sets of restraints across distances derived from the crystal structure of the antibody– epitope complex (set 1 and set 2 listed in Table S1). As expected, during SMD, the epitope started to move away from its initial conformation towards the conformation observed in the crystal structure of antibody–epitope complex with an associated decrease in rmsd (Figure 7) relative to the conformation in the crystal structure. The conformational changes at the epitope occurred at a slower rate under restraint set 1, as the former contained fewer numbers of restraints. The SMD simulations were carried out for 50ns and 30ns for set 1 and set 2 respectively. For the tethered EGFR–antibody complex system, the conformation of the epitope stabilized at < 5Å (Figure 7) under both restraints, albeit at different times. In contrast, in the untethered EGFR complex, while the epitope stabilized rapidly to < 5Å (Figure 7) under restraint set 2, it did not come closer than ~8Å (Figure 7) from the crystal structure under restraint set 1.


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Unrestrained molecular dynamics simulations The conformations obtained at the end of SMD were next subject to unrestrained MD simulations. The conformations of tethered EGFR–antibody at the end of SMD simulations under both sets of restraints and of untethered EGFR–antibody at the end of SMD simulations under restraint set 2 were quite stable (Figure 7). In contrast, the untethered EGFR–antibody conformation obtained at the end of SMD under restraint set 1 was relatively less stable (Figure 7), as the rmsd of sampled conformations fluctuated significantly (between 7– 12Å) during the unrestrained MD simulation; it is possible that longer simulations may lead to stability.

The binding energy of the antibody and the extracellular domain of EGFR and of the antibody and epitope (Figure 8) calculated over the conformations sampled during the unrestrained MD shows that the interaction energy is stronger for the full length extracellular domain than with the epitope alone. This is expected, as in EGFR, the antibody will be engaged in contacts with the epitope and with atoms that are outside this region. Binding energy calculations further reveal that the binding of antibody with the full-length extracellular domain of EGFR in its untethered state is more favorable than in the tethered state. The same pattern was observed when only the 16-residue epitope was considered. Moreover, the antibody also bound more strongly to the epitope from the untethered EGFR than in a control simulation starting from the antibodyepitope peptide crystal structure. These results agree with experimental observations that the antibody preferentially binds to the untethered conformations of EGFR.20 The conformation of the epitope from EGFR in its untethered state simulated under restraint set 1 is quite different from the conformation of the epitope in the crystal structure of the antibody – epitope complex (Figure 9).

This may reflect an initial recognition event, which subsequently adopts the



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conformations seen in the crystal structure and the other modes. Under restraint set 2, the average enthalpy of interaction of the antibody–EGFR complex was similar for both the untethered and tethered states and the interaction energies with the epitope in both states were similar to those in the crystal structure. Overall the antibody binding to the epitope in the context of full-length extracellular domain is mainly driven by favorable van der Waals and electrostatics interactions, with little contribution from nonpolar solvation energies.

Principal Component analysis Since the full-length extracellular domain of EGFR undergoes both local and global conformational changes during SMD to expose the buried epitope for antibody binding, principal component analysis (PCA) was carried out to quantify these conformational changes starting from the two different states of EGFR. PCA was performed on the conformations sampled during the last 10ns of the unrestrained simulations of the EGFR–antibody complex together with the simulation of the crystal structure of the antibody–epitope complex (Figure S5). Only the conformations of antibody and epitope extracted from the simulations of full length EGFR– antibody complexes were used. Distribution of PC1 and PC2 (Figure S5) shows that the conformations sampled during the untethered EGFR-antibody simulations (after using restraint set 1) occupied a distinct region of the PC1-PC2 plot that is quite different from the region sampled by the crystal structure. However, the conformations from the other simulations occupied regions around that of the crystal structure. This is consistent with the large rmsd (> 8Å) (Figure 7) values of the epitope sampled during the unrestrained simulation of the untethered EGFR-antibody complex under restraint set 1. PCA further revealed three distinct binding modes (Figure 10) for the antibody-epitope complex within the context of the full-length extracellular


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domain of EGFR. Binding modes 1 and 2 corresponds to conformations sampled during the unrestrained simulation of the untethered EGFR–antibody complex under restraint sets 1 and 2 respectively. Despite having similar antibody–epitope interaction energies (~ -50 kcal/mol), simulations of the untethered EGFR–antibody complex resulted in two different binding modes as a result of the two different sets of restraints. In contrast, both sets of restraints produced similar binding modes (binding mode 3) for the tethered EGFR, as evident from similarities in epitope RMSD (Figure 7), comparable average interaction energies (Figure 8) and their proximity in the PC1-PC2 plot (Figure S5). The binding mode (binding mode 3) that was observed for the binding of antibody to the tethered EGFR differs from the binding modes (modes 1 & 2) observed with untethered EGFR. In contrast to the previous computational studies19 where the peptide adopted the flipped-in conformation, we see the epitope in all three binding modes adopting a flipped-out conformation, which is consistent with the antibody– peptide crystal structure (Figure S6). We further evaluated the fraction of native contacts between the antibody and the epitope (Figure 11) and see that binding modes 2 and 3 reproduce most of the native contacts (~80%) thus lending further support to these modes of binding.

Free energy decomposition of the antibody–EGFR complex To determine the specific interactions that are important for structural stability and the overall binding energy, residue level decomposition analysis was performed on conformations sampled during unrestrained MD simulations of all the three binding modes. In addition to interaction energies, average number of hydrogen bonds, water-mediated hydrogen bonds and hydrophobic interactions for each epitope residue was calculated to identify residues that make important interactions with the antibody.



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Of the 16 epitope residues, C287 and C302 form a disulphide bond which stabilizes the loop. Residue level decomposition reveals that epitope residues G288, A289, D290, S291, Y292 and M294 do not interact appreciably with the antibody as evidenced by their negligible interaction energies (Figure 12), mirroring the available mutagenesis data (Table S2).21 We now focus on those residues that make significant contributions to the binding of the antibody.

In all the three binding modes, residues E293, E295, E296 (except binding mode 3), D297, G298, V299, R300 and K301, contribute ~10-15 kcal/mol to the interaction energies (Figure 12). Epitope residue E293 strongly interacts with the side chain of antibody residue Arg401 (Figure 13A). This salt bridge was quite stable during all 3 unrestrained simulations, consistent with results obtained from yeast display experiments showing decreased binding for the E293A/G/D mutations (Table S2). Our structural models (Figure 13) show that mutation of E293 to Ala, Gly and Asp will result in loss of the salt bridge, and will affect the stability. This is also consistent with abrogation of antibody binding when E293 was mutated to positively charged Lys.21 In binding modes 2 and 3, epitope residue E295 is involved in a network of interactions with residues Y354 from the antibody and R300 from the epitope (Figure 13B). However, in binding mode 1, the

overall conformation of the epitope positions these residues far apart, thus disabling the formation of such interactions. Instead, the side chain of epitope residue E295 interacts with the backbone of antibody residues A334 and G400 in binding mode 1 (Figure 13C). Such a side chain – main chain hydrogen bond interaction was observed in a majority of the conformations sampled during the simulation of the untethered EGFR–antibody complex (Figure S7). The reason why mutation of E295 does not appear to inhibit binding (Figure 12) may stem from the observation that E295 interacts only weakly with Y354 (of mAb806) and with R300 (of EGFR)


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(Figure 13B) in binding modes 2 and 3. However R300 makes a strong salt bridge with D332 (of mAb806) and is unlikely to get perturbed by the mutation of E295; indeed the Y354 side chain will probably reorient to recover its lost hydrogen bond with E295 through R300. In addition, the aliphatic carbon sidechain of E295 sits near a hydrophobic pocket formed by antibody residues F333, A334, W335, A339 and G400 (Figure 13) and mutation to Ala will thus promote interaction with the antibody.

Epitope residues E296 and D297 are also major contributing residues in binding modes 1, 2 and 3. Both the residues engage in a network of interactions with residues Y351, S353, R359 and N336 (Figure 13D and E) from the antibody through hydrogen bonds. In addition, residues E296 and D297 are involved in intermolecular interactions with K301 and K303 in binding modes1 and 2. Hence it is likely that mutation of either E296 or D297 alone will not destabilize local structural dynamics as the interaction with K301 will be stabilized by the remaining anion. This provides an explanation for the mutagenesis data (Table S2) showing that mutation of either E296 or D297 does not abrogate binding. Our current model then suggests that a double mutation (E296A/D297A) should affect binding.

Residues G298 and V299 both contribute ~7-10 kcal/mol to the interaction energies in all the three binding modes (Figure 12). Although these residues do not form strong interactions with the antibody, they are located in the flexible loop of the epitope region that buries into the pocket between the light and heavy chain of the antibody; this region is formed largely by hydrophobic residues (Y49, H50, Y91, W96, A399, Y351, W348) and negatively charged residues (E296, D297 N32) from the epitope and the antibody (Figure 13G). It is clear that mutations at these positions will likely destabilize the interactions with the antibody (Figure 13). Thus our model 19 John Wiley & Sons, Inc.


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rationalizes the available mutagenesis data whereby mutation of G298 and V299 to smaller residues such as Ala is well tolerated, while mutations to Ser and Asp are not, resulting in decreased binding affinity of the antibody.

Epitope residue R300, which is a major contributor in binding modes 2 and 3, makes moderate contributions in binding mode 1. It engages in a network of interactions with residues E293, E295 and D332. During the unrestrained simulations, water–mediated hydrogen bonds were observed between the epitope residue R300 and antibody residue R401 (Figure 13H & I). During the last 10 ns of the unrestrained simulations, the distance between the two arginines was ~ 5-7Å (Figure S8) for all the three different binding modes and in the simulation of the crystal structure of the antibody–epitope complex. Despite being involved in a stable salt bridge with D332, the experimental observation that mutation of R300 to Ala or Cys retains binding is surprising. One possible explanation could be that the R300-D332 salt bridge is exposed and hence contributes very little to the net electrostatic binding energy due to desolvation penalties, as has been suggested in other studies.40,41 The R300P mutation is likely to induce distortion in the epitope and hence may explain the abrogation of binding.

Epitope residue K301 forms numerous water-mediated hydrogen bonds (Figure S7) with the antibody. It is involved in intermolecular interactions with residues E296 and D297 in binding modes 1 & 2 and with antibody residues N32 and Y91 in binding mode 3, which are further involved in a network of interactions that stabilizes the binding of antibody to the epitope. Mutation to Glu will lead to electrostatic repulsion, thus destabilizing this region and hence


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attenuating binding, while mutation to Ala would remove the two hydrogen bonds, which would likely be compensated for by solvent molecules.

Structural changes in EGFR induced by the antibody Both the SMD and unrestrained simulations show that binding of antibody to the full-length extracellular domain of EGFR induced conformational changes in the epitope region as well in other regions. As is evident from the distance matrix (Cα) calculation of binding modes 1 and 2 observed from the simulation of the antibody–untethered EGFR complex, the presence of antibody caused an increase in distances between EGFR domains I and III, and between domains II and III (Figure S9). In contrast, these distances were observed to decrease in the simulations of the antibody–tethered EGFR complexes associated with binding mode 3. The global conformational changes in EGFR observed in our simulations in the presence of antibody, suggests/reveals that the untethered EGFR was being shifted closer to the tethered state by the antibody in binding modes 1 and 2, and the tethered EGFR shifted closer to the untethered state in binding mode 3, in accord with the suggestion that the antibody may initially recognize a conformation that is intermediate between the untethered and tethered states of EGFR.20 Furthermore, in all three binding modes, global conformational changes are coupled to local conformational changes at the epitope region in EGFR, that clearly are induced by antibody binding (21). Increase in epitope rmsd with respect to its conformation in the uncomplexed state together with increases in distances between the epitope and domain II was observed in all the simulations, (Figure S9), clearly reflecting antibody-induced local unfolding of the epitope.

In all our binding models, in addition to the interactions with the epitope residues, the antibody also forms stable interactions with other regions of EGFR (Figure S10). In particular, in binding 21 John Wiley & Sons, Inc.


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modes 1 and 2, mAb806 interacts with the dimerization arm (residues 242-259) of the EGFR in domain II. This may serve to inhibit receptor dimerization or oligomerization, thus preventing receptor activation, a mechanism that is thought to characterize the inhibition of EGFR by the antibody pertuzumab.42

Discussion Most biological processes in cells are driven by protein–protein interactions. They modulate the amplitude and duration of cellular signaling pathways. These interactions are often associated with conformational changes or allosteric changes.43 EGFR is one such protein, where dimerization–driven activation of the intracellular kinase domain is dependent upon extracellular ligand binding and is crucial to cellular pathways regulating proliferation, migration and differentiation. Due to the aberrant activation of this receptor in several cancers, its inhibition by either targeting the extracellular domains using monoclonal antibodies (mAb) or the intracellular kinase domain using small molecule kinase inhibitors has been successfully exploited therapeutically. While a significant amount is known regarding the latter, relatively little is known about the antibody binding/inhibition mechanisms. Antibodies act as allosteric inhibitors of enzymes and receptors, targeting different regions of proteins, often involving multiple regions of the protein that are sequentially disjointed (several examples are highlighted later). For instance, the antibody Pertuzumab binds to the ErbB2 receptor near the center of extracellular domain II to prevent ligand-induced dimerization of ErbB2 with other ErbB receptors, while the antibody Trastuzumab (Herceptin) binds to the juxtamembrane region of the HER2 (ErbB2) receptor to inhibit its activation.44 In addition, these two antibodies can synergize to inhibit HER2 activation.45 22 John Wiley & Sons, Inc.

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A newly discovered monoclonal antibody mAb806 was found to bind to a truncated form of the EGFR present in some cancers. This antibody was later also found to bind to over-expressed wild–type EGFR on cancer cells, surprisingly sparing wild type EGFR in normal cells. Glycosylation ahs been invoked as particular underlying mechanism, but unequivocal evidence is lacking. Nevertheless the differential binding ability of the antibody represents a promising development of designing therapeutics with high specificity and hence understanding its mechanism is crucial. Epitope mapping studies show that mAb806 recognizes a disulphidebonded loop in EGFR as was further demonstrated in a crystal structure of the antibody complexed with this peptide fragment. While this epitope is constitutively exposed in the (∆27EGFR) mutant, explaining the selective binding of mAb806 to the truncated EGFR, it is not easily accessible in the full-length extracellular domain (in both the tethered and untethered states). Thus available structural models are unable to rationalize the selective binding of antibody to wild type EGFR. In this study we have used various computational approaches to provide a structural model for the binding of antibody to the epitope in the full-length extracellular domain of EGFR.

Simple docking the crystal structure of the antibody to the wild type EGFR in its tethered and untethered states fails to demonstrate a close approach between the antibody and the cryptic epitope region. This was not unexpected as incorporation of large conformational changes, such as induced fit etc, are still not realized in current docking algorithms.46 Hence we next we carried out classical MD simulations of the docked EGFR–antibody complexes, but these too failed to capture the fluctuations necessary for the epitope to be exposed sufficiently for recognition by the antibody. Again, this was not really surprising given the short timescales being simulated.



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Then we resorted to SMD, a biasing method that utilizes time-dependent external forces to induce structural changes in biomolecules.47,48 This method has been used successfully to study ligand binding or unbinding pathways49,50 but applications to large systems such as antibody– antigen complexes are sparse. In the current work, SMD was carried out under distance restraints that would bring a group of residues into proximity. These are residues in the antibody and the epitope that have been shown crystallographically to interact with each other. SMD successfully steered the system such that the epitope-antibody complex closely resembled (rmsd ~ 3 Å) the crystal structure. These conformations were further subject to unrestrained MD simulations over 50ns (data not shown) and were found to be stable.

Principal component analysis of the sampled conformations yielded three different antibody– epitope binding modes. The two different sets of distance restraints resulted in the same binding mode for the antibody-tethered complex, whereas different binding modes were observed in the case of the antibody–untethered model. Binding energies during the unrestrained simulations of the antibody–EGFR complex in the three different binding modes revealed that the antibody binds more favorably to the untethered EGFR than to the tethered EGFR, as supported by experiments.20 Interestingly the antibody also bound as strongly to the epitope from untethered EGFR as in a control simulation of the antibody-epitope peptide crystal structure; however the conformation of the epitope from the antibody bound to full-length EGFR in its untethered state is somewhat different form that seen in the crystal structures of the antibody–epitope complex. This could reflect an initial recognition event that is followed by conformational adaptations that lead to the states seen in the crystal structure and in the other modes. It is also possible that longer simulations would show convergence in this mode to the crystal structure conformation.


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Nevertheless the 3 binding modes identified from this study can individually or together lend support to the growing appreciation that protein-protein, or protein-ligand interactions are best described by conformational ensembles consisting of multiple binding modes.51

Detailed analysis of the unrestrained EGFR–antibody MD simulations revealed several residues to be important for the different binding modes. These are in accord with the idea that binding hotspots, as determined from mutagenesis data, lie on the binding interfaces, and contribute significantly to the experimentally observed binding affinity. Most of the epitope residues including E293, E295, E296, D297, G298, V299, R300 and K301 contribute significantly to the binding energies and play key roles in stabilizing the bound conformation of the antibody with the epitope. Our structural models suggest that the lack of effect of mutating certain epitope residues individually to Ala may result from the presence of several charged residues in this neighborhood that could easily re-orient to form compensatory interactions with the antibody. This possibility can be examined by the use of double or triple Ala scans of the EGFR/epitope residues.52 Of course, there are key interactions where perturbation, by Ala mutations, of electrostatics, van der Waals and hydrophobic interactions are not tolerated and result in the observed attenuation of antibody binding to EGFR.

In summary, we have used computational docking combined with biased and unbiased MD simulations to model and study possible modes of complexation between the antibody mAb806 and models of the full-length extracellular domain of EGFR. Our modeling study finds that the antibody mAb806 appears to bind most optimally to a conformation of EGFR that is in a state intermediate between tethered and untethered EGFR and is coupled with both local unfolding of the epitope and global conformational changes in EGFR. Our structural models are largely able 25 John Wiley & Sons, Inc.


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to rationalize the available experimental and mutagenesis data. In addition, our structural models further suggest that the antibody makes additional contacts with the dimerization arm of EGFR and may serve to inhibit dimerization. While detailed examination of the current structural models do not show direct involvement of any glycosylation sites in antibody binding,14,53 N579 is in close proximity to the antibody binding interface in binding mode 1 (Figure S11).19 The structural models generated in this study can serve as templates for designing potent ligands or antibody therapies targeting transiently exposed epitopes with increased specificity. Moreover the methodology adopted in this study can easily be used to model the interaction of large biomolecules with hidden binding sites, which require local unfolding and or large conformational changes for binding.

A key finding from such studies is that the traditional view of a single continuous epitope eliciting binding from an antibody must be revised by expanding the definition to one that includes multiple discontinuous epitopes, with perhaps one dominant epitope. It is not surprising then that the associated dynamically fluctuating surfaces must lead to the exposure transiently of sites that are otherwise cryptic. These observations indeed are not new and the following are some examples of the spectrum of such interactions: gp120 of HIV-1 unveils hidden epitopes in CD4 on the human membrane, leading to T-cell activation;54,

55

cryptic epitopes have been

hypothesized to often emerge under conditions of high concentrations, leading to T lymphocyte activation/recognition56 as has been suggested to explain the unusual efficacy of mAb806 in cancer cells, where EGFR concentrations are known to be very high compared to normal cells; antibodies against human growth hormones recognize cryptic eptiopes that are discontinuous;57 cryptic epitopes have been targeted in tumour immunotherapy;58 an antibody against the C-Met receptor has recently been shown to be temperature sensitive and recognizes a buried epitope that 26 John Wiley & Sons, Inc.

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is presented across a disulphide bridge,59 as in the current study of EGFR; the antibodies rituximab and atumumab are classified as Type I antibodies against CD20 but recognize different epitopes while tositumumab which is a Type II antibody, recognizes an epitope that is similar to that recognized by rituximab.60 Indeed rituximab has been shown recognize a loop that is presented by a disluphide (as is the case for C-Met and EGFR), together with a discontinuous motif (curiously cyclic peptides that present the major epitope on a loop are recognized by rituximab, but linear peptides with very different sequences are also recognized by rituximab); the CemX domain of the membrane bound IgE is the target for the development of therapeutic antibodies for allergies,61 with both antibodies targeting an intrinsically disordered region of CemX, yet binding to different conformations. Finally, Panitumumab and Cetuximab, both target EGFR62 at regions that are distinct from that targeted by mAb806: Cetuximab, a chimeric mouse/human antibody recognizes a large spatial region of domain III in EGFR which is made up of discontinuous epitopes; Panitumumab, a fully human antibody also targets discontinuous regions of domain III of EGFR, some of which overlap with those targeted by Cetuximab. Herceptin or Transtuzumab binds to the juxtamembrane region of the extracellular part of the Her2 receptor44 engaging a series of residues from sequentially disparate regions while Pertuzumab targets a large region near domain 2 of Her263 engaging several disconnected surfaces including a loop region that is presented by a disulphide bridge; a targeted biologic, adnectin, derived from human immunoglobulin fibronectin, interacts with domain 1 of EGFR across multiple disconnected epitopes26. Clearly this list is not exhaustive but serves to establish that the forces that mediate antibody-antigen complexation are composed of several subtle interactions. These interactions arise from the underlying dynamically fluctuating surfaces which combine some regions with low flexibility that may be termed as “major eptiopes” and several



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other regions that are sequentially disparate but brought into spatial contiguity as a consequence of folding. It is not surprising that some of these regions may ordinarily appear to be cryptic from structural studies, but under the modulation of approaching partners or environmental conditions, be transiently exposed to enable fine-tuning and hence make possible the diverse range of exquisite specificities that govern antibody recognition. Indeed, some of these studies have also demonstrated that these differential interactions are associated with differing conformations and orientations of the antibodies which has implications for subsequent signaling that recruits the complement system and/or the ADCC system,64 both critical for eliciting a desired biological response.

Acknowledgement The authors thank A*STAR Computing Resource Centre (A*CRC) for computing facilities.

Supporting Information Attached separately.


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Figure Legends Figure 1:

Crystal structure of the antibody–epitope complex (PDB id 3G5V). Cartoon

representation of the Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green) are shown. (B) The eptiope shown in cartoon with the residues in stick representation. (C) The interactions between the antibody and the epitope, with all the interacting residues shown as sticks. Figure 3: Backbone RMSD of the EGFR conformations tethered (black) and untethered (red) sampled during the simulations with Domain IV restrained. The RMSD was calculated after aligning the complete extracellular domain of EGFR from each frame of the trajectory to the crystal structure. Figure 3: Backbone RMSD of the conformations of the antibody (black) and epitope (red) sampled during the simulations. The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure. Figure 4: Different binding modes (clusters) produced by docking mAb806 to the untethered and tethered EGFR using HADDOCK. The extracellular domains of the EGFR (residues 1-612) are depicted in surface representation and the antibody is shown in cartoon. The epitope region is colored green and the light and heavy chains of the antibody are in orange and red respectively. The HADDOCK score and RMSD from the best model are plotted for each cluster member. HADDOCK score = Van der Waals energy (EVDW) * 1.0 + Electrostatic energy (EELE) * 0.2 + Desolvation energy (ESOLV) * 1.0 + Restraint violation energy (EAIR) * 0.1. Cluster averages are shown as black dots with error bars indicating one standard deviation from the mean.


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Figure 5: Antibody docking to the extracellular domain of EGFR. Conformation of the epitope (magenta) in the docked models of full length extracellular domain of EGFR untethered (A) and tethered (B) with antibody was quite different from the conformation of the epitope observed in the crystal structure of the antibody–epitope complex. Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green). Figure 6: Backbone RMSD of the conformations of antibody (A) and epitope (B &C) sampled during the simulations of antibody – EGFR complex with EGFR in its tethered (red) and untethered (black) states against its conformation in the crystal structure of antibody-epitope complex (PDB id 3G5V) (A & C) and EGFR (B). The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure. Figure 7: Backbone RMSD of the conformations of epitope sampled during the SMD simulations (A,C) with restraint set 1 (A) and set 2 (C) of antibody – EGFR complex with EGFR in its untethered (red) and tethered (black) states against its conformation in the crystal structure of antibody-epitope complex. (B, D) Backbone RMSD of the conformations of epitope sampled during unrestrained simulations following the SMD simulations with restraint set 1 (B) and set 2 (D) of antibody – EGFR complex with EGFR in its untethered (red) and tethered (black) states against its conformation in the crystal structure of antibody-epitope complex. The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure. Figure 8: Average enthalpy of interaction upon removal of all restraints. 20 ns of unrestrained MD simulations were carried out after 50 ns of steered MD for restraint set 1 and 30 ns for restraint set 2.

The average interaction energy was calculated over the last 10 ns of the

unrestrained simulations. The average interaction energy of the control was calculated from the



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last 10 ns of a 20 ns MD simulation starting from the crystal structure. Untethered with restraint set 1 refers to binding mode 1; untethered with restraint set 2 refers to binding mode 3; tethered with restraint sets 1 and 2 refer to binding mode 2. Figure 9: Conformation of the epitope (magenta) after SMD simulations with set 1 (A, C) and set 2 ( B, D) of full length extracellular domain of EGFR untethered (C, D) and tethered (A, B) with the epitope observed in the crystal structure of the antibody–epitope complex. Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green).

Figure 10: Binding modes obtained after steered MD and 20 ns of unrestrained simulations. (A) Binding mode 1 corresponds to untethered EGFR under restraint set 1; (B) binding mode 2 corresponds to untethered EGFR under restraint set 2; (C) binding mode 3 corresponds to tethered EGFR under restraint set 1 and 2. The epitope is colored magenta, antibody heavy chains in cyan and antibody light chains in grey. The epitope from the crystal structure is shown in green. Peptide backbones are shown in cartoon representation and interacting side chains as sticks. The view is that of looking into the antigen binding site of the antibody. Epitope residues are labeled in bold. Antibody heavy chain residue numbers are altered (add 300). Figure 11: Evolution of fraction of native binding contacts. Presence of native binding contacts between the epitope – antibody conformations sampled during the simulations of (black) antibody – epitope crystal structure (red) binding mode 1 (blue) binding mode 2 (green) binding mode 3. Figure 12: Contribution of each EGFR epitope residue to interaction energy with the antibody. Each point represents the average interaction energy over the final 10 ns of the unrestrained MD


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simulation. The average interaction energies for the control were calculated between 10 ns and 25 ns (the stable portion) of the MD simulation of the crystal structure (PDB: 3G5V). Error bars represent one standard deviation from the mean. For binding mode 3, the unrestrained simulation for the tethered EGFR after restraint set 1 was used for calculations. Figure 13: Close up view of epitope residue interactions. All figures were obtained from binding mode 1, 2 or 3 after steered MD and 20 ns of unrestrained simulations. The epitope is colored pink and the rest of the EGFR in light blue; Antibody heavy chains are in green and light chains in orange.



Figure 1 Crystal structure of the antibody–epitope complex (PDB id 3G5V). Cartoon representation of the Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green) are shown. (B) The eptiope shown in cartoon with the residues in stick representation. (C) The interactions between the antibody and the epitope, with all the interacting residues shown as sticks.

John Wiley & Sons, Inc.

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Figure 2 Backbone RMSD of the EGFR conformations tethered (black) and untethered (red) sampled during the simulations with Domain IV restrained. The RMSD was calculated after aligning the complete extracellular domain of EGFR from each frame of the trajectory to the crystal structure



Figure 3 Backbone RMSD of the conformations of the antibody (black) and epitope (red) sampled during the simulations. The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure


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Figure 4 Different binding modes (clusters) produced by docking mAb806 to the untethered and tethered EGFR using HADDOCK. The extracellular domains of the EGFR (residues 1-612) are depicted in surface representation and the antibody is shown in cartoon. The epitope region is colored green and the light and heavy chains of the antibody are in orange and red respectively. The HADDOCK score and RMSD from the best model are plotted for each cluster member. HADDOCK score = Van der Waals energy (EVDW) * 1.0 + Electrostatic energy (EELE) * 0.2 + Desolvation energy (ESOLV) * 1.0 + Restraint violation energy (EAIR) * 0.1. Cluster averages are shown as black dots with error bars indicating one standard deviation from the mean.



Figure 5 Antibody docking to the extracellular domain of EGFR. Conformation of the epitope (magenta) in the docked models of full length extracellular domain of EGFR untethered (A) and tethered (B) with antibody was quite different from the conformation of the epitope observed in the crystal structure of the antibody–epitope complex. Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green).


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Figure 6 Backbone RMSD of the conformations of antibody (A) and epitope (B &C) sampled during the simulations of antibody – EGFR complex with EGFR in its tethered (red) and untethered (black) states against its conformation in the crystal structure of antibody-epitope complex (PDB id 3G5V) (A & C) and EGFR (B). The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure.



Figure 7 Backbone RMSD of the conformations of epitope sampled during the SMD simulations (A,C) with restraint set 1 (A) and set 2 (C) of antibody – EGFR complex with EGFR in its untethered (red) and tethered (black) states against its conformation in the crystal structure of antibody-epitope complex. (B, D) Backbone RMSD of the conformations of epitope sampled during unrestrained simulations following the SMD simulations with restraint set 1 (B) and set 2 (D) of antibody – EGFR complex with EGFR in its untethered (red) and tethered (black) states against its conformation in the crystal structure of antibody-epitope complex. The RMSD was calculated after aligning the antibody from each frame of the trajectory to the crystal structure.


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Figure 8 Average enthalpy of interaction upon removal of all restraints. 20 ns of unrestrained MD simulations were carried out after 50 ns of steered MD for restraint set 1 and 30 ns for restraint set 2. The average interaction energy was calculated over the last 10 ns of the unrestrained simulations. The average interaction energy of the control was calculated from the last 10 ns of a 20 ns MD simulation starting from the crystal structure. Untethered with restraint set 1 refers to binding mode 1; untethered with restraint set 2 refers to binding mode 3; tethered with restraint sets 1 and 2 refer to binding mode 2.



Figure 9 Conformation of the epitope (magenta) after SMD simulations with set 1 (A, C) and set 2 ( B, D) of full length extracellular domain of EGFR untethered (C, D) and tethered (A, B) with the epitope observed in the crystal structure of the antibody–epitope complex. Light chains (cyan) and heavy chains (Grey) of the antibody and the EGFR epitope (green).


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Figure 10 Binding modes obtained after steered MD and 20 ns of unrestrained simulations. (A) Binding mode 1 corresponds to untethered EGFR under restraint set 1; (B) binding mode 2 corresponds to untethered EGFR under restraint set 2; (C) binding mode 3 corresponds to tethered EGFR under restraint set 1 and 2. The epitope is colored magenta, antibody heavy chains in cyan and antibody light chains in grey. The epitope from the crystal structure is shown in green. Peptide backbones are shown in cartoon representation and interacting side chains as sticks. The view is that of looking into the antigen binding site of the antibody. Epitope residues are labeled in bold. Antibody heavy chain residue numbers are altered (add 300).



Figure 11 Evolution of fraction of native binding contacts. Presence of native binding contacts between the epitope – antibody conformations sampled during the simulations of (black) antibody – epitope crystal structure (red) binding mode 1 (blue) binding mode 2 (green) binding mode 3.


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Figure 12 Contribution of each EGFR epitope residue to interaction energy with the antibody. Each point represents the average interaction energy over the final 10 ns of the unrestrained MD simulation. The average interaction energies for the control were calculated between 10 ns and 25 ns (the stable portion) of the MD simulation of the crystal structure (PDB: 3G5V). Error bars represent one standard deviation from the mean. For binding mode 3, the unrestrained simulation for the tethered EGFR after restraint set 1 was used for calculations.



Figure 13 Close up view of epitope residue interactions. All figures were obtained from binding mode 1, 2 or 3 after steered MD and 20 ns of unrestrained simulations. The epitope is colored pink and the rest of the EGFR in light blue; Antibody heavy chains are in green and light chains in orange.


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Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

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Nonradioactive ligand binding assay for epidermal growth factor receptor.

Binding of epidermal growth factor to its receptor is affected by membrane phospholipid environment.

Adrenal cortical neoplasms: a study of clinicopathological features related to epidermal growth factor receptor gene status.

Epidermal growth factor receptor in pancreatic cancer.

Epidermal growth factor receptor in glioblastoma.

Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity.

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

Cutaneous adverse reactions specific to epidermal growth factor receptor inhibitors.

Direct linkage of epidermal growth factor to its receptor.

Decrease in epidermal growth factor receptor levels and production of material enhancing epidermal growth factor binding accompany the temperature-dependent changes from normal to transformed phenotype.

Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis.

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor.

Growth fractions of breast cancer in relation to epidermal growth factor receptor and estrogen receptor.

Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study.

Cytokine regulation by epidermal growth factor receptor inhibitors and epidermal growth factor receptor inhibitor associated skin toxicity in cancer patients.

Oncogenic fingerprint of epidermal growth factor receptor pathway and emerging epidermal growth factor receptor blockade resistance in colorectal cancer.

Expression of epidermal growth factor receptor and human epidermal growth factor receptor 2 in periocular squamous cell carcinomas of horses.

Epidermal growth factor receptor inhibitors: a patent review (2010 - present).

Inhibiting epidermal growth factor receptor at a distance.

Epidermal growth factor receptor and bladder cancer: a review.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

Scintillation proximity assay to study the interaction of epidermal growth factor with its receptor.