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Capturing transient antibody conformations with DNA origami epitopes.

ABSTRACT: Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.

SUBMITTER: Zhang P

PROVIDER: S-EPMC7305102 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Capturing transient antibody conformations with DNA origami epitopes.

Zhang Ping P Liu Xiaoguo X Liu Pi P Wang Fei F Ariyama Hirotaka H Ando Toshio T Lin Jianping J Wang Lihua L Hu Jun J Li Bin B Fan Chunhai C

Nature communications 20200619 1

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immu ...[more]

PMID: 32561744

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Project description:The human DNA repair enzyme uracil DNA glycosylase (hUNG) locates and excises rare uracil bases that arise in DNA from cytosine deamination or through dUTP incorporation by DNA polymerases. Previous NMR studies of hUNG have revealed millisecond time scale dynamic transitions in the enzyme-nonspecific DNA complex, but not the free enzyme, that were ascribed to a reversible clamping motion of the enzyme as it scans along short regions of duplex DNA in its search for uracil. Here we further probe the properties of the nonspecific DNA binding surface of {(2)H(12)C}{(15)N}-labeled hUNG using a neutral chelate of a paramagnetic Gd(3+) cosolute (Gd(HP-DO3A)). Overall, the measured paramagnetic relaxation enhancements (PREs) on R(2) of the backbone amide protons for free hUNG and its DNA complex were in good agreement with those calculated based on their relative exposure observed in the crystal structures of both enzyme forms. However, the calculated PREs systematically underestimated the experimental PREs by large amounts in discrete regions implicated in DNA recognition and catalysis: active site loops involved in DNA recognition (268-274, 246-250), the uracil binding pocket (143-148, 169-170), a transient extrahelical base binding site (214-216), and a remote hinge region (129-132) implicated in dynamic clamping. These reactive hot spots were not correlated with structural, hydrophobic, or solvent exchange properties that might be common to these regions, leaving the possibility that the effects arise from dynamic sampling of exposed conformations that are distinct from the static structures. Consistent with this suggestion, the above regions have been previously shown to be flexible based on relaxation dispersion measurements and course-grained normal-mode analysis. A model is suggested where the intrinsic dynamic properties of these regions allows sampling of transient conformations where the backbone amide groups have greater average exposure to the cosolute as compared to the static structures. We conclude that PREs derived from the paramagnetic cosolute reveal dynamic hot spots in hUNG and that these regions are highly correlated with substrate binding and recognition.

Dataset Information

Capturing transient antibody conformations with DNA origami epitopes.

Publications

Capturing transient antibody conformations with DNA origami epitopes.

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