Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES

Yuji Kado,Eiichi Mizohata,Satoru Nagatoishi,Mariko Iijima,Keiko Shinoda,Takamitsu Miyafusa,Taisuke Nakayama,Takuma Yoshizumi,Akira Sugiyama,Takeshi Kawamura,Young-Hun Lee,Hiroyoshi Matsumura,Hirofumi Doi,Hideaki Fujitani,Tatsuhiko Kodama,Yoshikazu Shibasaki,Kouhei Tsumoto,Tsuyoshi Inoue

doi:10.1074/jbc.m115.656009

Yuji Kado, Eiichi Mizohata + Show 16 more

Open Access

https://doi.org/10.1074/jbc.m115.656009

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Abstract

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro103. A molecular dynamics simulation and mutational analyses revealed that Arg40 in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Highlights

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells
Instance, trastuzumab is a humanized IgG1␬ monoclonal antibody that is targeted for the human epidermal growth factor (EGF) receptor (EGFR)6 2 (HER2, ErbB-2), which is used in the treatment of metastatic breast cancer [1]
The interaction between 9E5(Fab) and hEPR formed a solvent-accessible surface of ϳ919 Å2, which is in the typical range of interaction surfaces between antibodies and antigens [32]

Summary

Experimental Procedures

Production and Purification of 9E5(Fab)—The 9E5 monoclonal antibody was produced using a method described previously [11]. For x-ray data collection, a 9E5(Fab) crystal was soaked in cryoprotectant solution (50 mM HEPES-Na (pH 7.3), 24% (v/v) PEG 4000, and 10% (v/v) glycerol) and flash frozen in liquid nitrogen. For crystallization of the 9E5(Fab)1⁄7hEPR complex, 0.5 ␮l of protein solution (10 mg mlϪ1 9E5(Fab)1⁄7hEPR, 20 mM Tris-HCl (pH 7.5), and 300 mM NaCl) was mixed with 0.5 ␮l of reservoir solution (100 mM MES monohydrate (pH 6.0) and 14% (v/v) PEG 4000) at 20 °C. A 9E5(Fab)1⁄7hEPR crystal was soaked in cryoprotectant solution (100 mM MES monohydrate (pH 6.0), 17% (w/v) PEG 4000, and 20% (v/v) glycerol) and flash frozen in liquid nitrogen. The structure of 9E5(Fab) was determined by the molecular replacement method using 82D6A3, which is an antithrombotic antibody [15] (Protein Data Bank code 2ADF), as the starting model with PHASER [16]. Four NPT (constant number of particles, pressure, and temperature) simulations were performed for 1 s with initial random velocities that obeyed a Maxwell-Boltzmann distribution at 298 K

No of unique reflections

Residue Atom Residue CDR Atom distance

Results

Calculation of the Interaction Energy by Molecular

EPR Residue CDR

Discussion