title: Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.
abstract: Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Takamitsu
authors: T
authors: Hattori
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Darson
authors: D
authors: Lai
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Irina S
authors: IS
authors: Dementieva
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Sherwin P
authors: SP
authors: Montaño
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Kohei
authors: K
authors: Kurosawa
authors: Department of Chemistry, Northwestern University, Evanston, IL 60208; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208;
authors: Yupeng
authors: Y
authors: Zheng
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Louesa R
authors: LR
authors: Akin
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, 50-370, Wrocław, Poland;
authors: Kalina M
authors: KM
authors: Świst-Rosowska
authors: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637;
authors: Adrian T
authors: AT
authors: Grzybowski
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637;
authors: Akiko
authors: A
authors: Koide
authors: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
authors: Krzysztof
authors: K
authors: Krajewski
authors: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
authors: Brian D
authors: BD
authors: Strahl
authors: Department of Chemistry, Northwestern University, Evanston, IL 60208; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208;
authors: Neil L
authors: NL
authors: Kelleher
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637;
authors: Alexander J
authors: AJ
authors: Ruthenburg
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; skoide@uchicago.edu.
authors: Shohei
authors: S
authors: Koide
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000941:Antigens / Q000737:chemistry* / Q000235:genetics; D001666:Binding Sites, Antibody; D018360:Crystallography, X-Ray; D006657:Histones / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D008745:Methylation; D020836:Protein Structure, Quaternary
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D006657:Histones; D007140:Immunoglobulin Fab Fragments
keywords: antibody engineering; antibody validation; data reproducibility; epigenetics; protein–protein interaction