Unveiling the structural mechanisms behind high affinity and selectivity in phosphorylated epitope-specific rabbit antibodies

Keisuke Kasahara; Raiji Kawade; Makoto Nakakido; Ryo Matsunaga; Hiroki Akiba; Kevin C Entzminger; Toshiaki Maruyama; Shigeru C J Okumura; Jose M M Caaveiro; Daisuke Kuroda; Kouhei Tsumoto

doi:10.1016/j.jbc.2024.107989

Unveiling the structural mechanisms behind high affinity and selectivity in phosphorylated epitope-specific rabbit antibodies

J Biol Chem. 2024 Nov 13;300(12):107989. doi: 10.1016/j.jbc.2024.107989. Online ahead of print.

Authors

Affiliations

¹ Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
² Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
³ Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki City, Osaka, Japan; Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
⁴ Abwiz Bio Inc., San Diego, California, USA.
⁵ Laboratory of Protein Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka-shi, Japan.
⁶ Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan. Electronic address: [email protected].
⁷ Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki City, Osaka, Japan; Abwiz Bio Inc., San Diego, California, USA; Medical Proteomics Laboratory, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan. Electronic address: [email protected].

PMID: 39542251
DOI: 10.1016/j.jbc.2024.107989

Abstract

Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a monophosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of posttranslational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

Keywords: antibody; antigen; molecular dynamics; peptide interaction; posttranslational modification; protein structure.