title: Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.
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 Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) 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.
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan, Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kado
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Mizohata
authors.affiliation: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku Tokyo 108-8639, Japan, and.
authors.forename: Satoru
authors.initials: S
authors.lastname: Nagatoishi
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Iijima
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Shinoda
authors.affiliation: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku Tokyo 108-8639, Japan, and.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Miyafusa
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Nakayama
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.
authors.forename: Takuma
authors.initials: T
authors.lastname: Yoshizumi
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sugiyama
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawamura
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Young-Hun
authors.initials: YH
authors.lastname: Lee
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Doi
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Fujitani
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Number 34 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Shibasaki
authors.affiliation: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku Tokyo 108-8639, Japan, and tsumoto@ims.u-tokyo.ac.jp.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.affiliation: From the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan, inouet@chem.eng.osaka-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Inoue
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000276:immunology; D002151:Calorimetry; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D066260:Epiregulin / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C587880:EREG protein, human; D066260:Epiregulin; D007074:Immunoglobulin G; D011994:Recombinant Proteins
keywords: antibody; cancer; crystal structure; epidermal growth factor (EGF); molecular dynamics; thermodynamics