Dataset Information

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.

SUBMITTER: Rajagopalan K

PROVIDER: S-EPMC39181 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Novel unconventional binding site in the variable region of immunoglobulins.

Rajagopalan K K Pavlinkova G G Levy S S Pokkuluri P R PR Schiffer M M Haley B E BE Kohler H H

Proceedings of the National Academy of Sciences of the United States of America 19960601 12

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 ...[more]

PMID: 8650212

Similar Datasets

Project description:Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.

Dataset Information

Novel unconventional binding site in the variable region of immunoglobulins.

Publications

Novel unconventional binding site in the variable region of immunoglobulins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure