title: Novel unconventional binding site in the variable region of immunoglobulins.
abstract: The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.
authors.affiliation: Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, Stanford University School of Medicine, CA 94305, USA.
authors.forename: K
authors.initials: K
authors.lastname: Rajagopalan
authors.forename: G
authors.initials: G
authors.lastname: Pavlinkova
authors.forename: S
authors.initials: S
authors.lastname: Levy
authors.forename: P R
authors.initials: PR
authors.lastname: Pokkuluri
authors.forename: M
authors.initials: M
authors.lastname: Schiffer
authors.forename: B E
authors.initials: BE
authors.lastname: Haley
authors.forename: H
authors.initials: H
authors.lastname: Kohler
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid
chemical_list: D000345:Affinity Labels; D007135:Immunoglobulin Variable Region