Single-Molecule 3D Images of "Hole-Hole" IgG1 Homodimers by Individual-Particle Electron Tomography.

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TitleSingle-Molecule 3D Images of "Hole-Hole" IgG1 Homodimers by Individual-Particle Electron Tomography.
Publication TypeJournal Article
Year of Publication2019
AuthorsLei, D, Liu, J, Liu, H, Cleveland, TE, Marino, JP, Lei, M, Ren, G
JournalSci Rep
Volume9
Issue1
Pagination8864
Date Published2019 Jun 20
ISSN2045-2322
Abstract

The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.

DOI10.1038/s41598-019-44978-7
Alternate JournalSci Rep
PubMed ID31221961
Grant ListDE-AC02-05CH11231 / / DOE | Office of Science (SC) /
R01HL115153 / / U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI) /
R01GM104427 / / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) /