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Structural basis for the recognition of C20:2-?GalCer by the invariant natural killer T cell receptor-like antibody L363.

ABSTRACT: Natural killer T (NKT) cells express a semi-invariant V?14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to ?- and ?-anomeric glycosphingolipids, as well as microbial ?-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen ?GalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 ? resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the ?GalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented ?GalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3? residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F' roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.

SUBMITTER: Yu ED

PROVIDER: S-EPMC3256878 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Structural basis for the recognition of C20:2-αGalCer by the invariant natural killer T cell receptor-like antibody L363.

Yu Esther Dawen ED Girardi Enrico E Wang Jing J Mac Thien-Thi TT Yu Karl O A KO Van Calenbergh Serge S Porcelli Steven A SA Zajonc Dirk M DM

The Journal of biological chemistry 20111122 2

Natural killer T (NKT) cells express a semi-invariant Vα14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to α- and β-anomeric glycosphingolipids, as well as microbial α-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen αGalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidat ...[more]

PMID: 22110136

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Project description:BACKGROUND:Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. METHODS:The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). RESULTS:A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. CONCLUSIONS:The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

Dataset Information

Structural basis for the recognition of C20:2-?GalCer by the invariant natural killer T cell receptor-like antibody L363.

Publications

Structural basis for the recognition of C20:2-αGalCer by the invariant natural killer T cell receptor-like antibody L363.

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