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

Published

June 20, 2019

Author(s)

Dongsheng Lei, Jianfang Liu, Hongbin Liu, Thomas Cleveland, John Marino, Ming Lei, Gang Ren

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 the three- dimensional (3D) structure of the synthetic IgG. However, due to the flexible nature of these molecules, their structure determination 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 species is an undesired variant generated during the production of a bispecific antibody using the knob- into-hole heterodimer technology. A total of sixty-four 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.

Citation

Scientific Reports

Volume

9

Issue

1

Pub Type

Journals

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DOI Link

Keywords

Electron Tomography, Electron Microscopy, EM, CryoEM, Immunoglobulin G, IgG, NISTmAb, IPET, Individual Particle Electron Tomography, Monoclonal Antibody, mAb

Citation

Lei, D. , Liu, J. , Liu, H. , Cleveland, T. , Marino, J. , Lei, M. and Ren, G. (2019), Single-Molecule 3D Imaging of “Hole-Hole” IgG1 Homodimers by Individual-Particle Electron Tomography, Scientific Reports, [online], https://doi.org/10.1038/s41598-019-44978-7 (Accessed April 19, 2024)