Single-Molecule 3D Images of 'Hole-Hole' IgG1 Homodimers by Individual-Particle Electron Tomography
| Autor: | Dongsheng Lei, Hongfang Liu, John P. Marino, Gang Ren, Ming Lei, Jianfang Liu, Thomas E. Cleveland |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Bispecific antibody Electron Microscope Tomography Protein Conformation Molecular imaging lcsh:Medicine Negative Staining Article Antibodies Imaging 03 medical and health sciences Imaging Three-Dimensional 0302 clinical medicine Protein structure Antibodies Bispecific Electron microscopy Molecule lcsh:Science Multidisciplinary Chemistry lcsh:R Negative stain 3. Good health Characterization (materials science) Molecular Imaging Other Physical Sciences Fc domain 030104 developmental biology Electron tomography Immunoglobulin G Three-Dimensional Biophysics Particle lcsh:Q Bispecific Biochemistry and Cell Biology Protein design Protein Multimerization 030217 neurology & neurosurgery |
| Zdroj: | Scientific reports, vol 9, iss 1 Scientific Reports, Vol 9, Iss 1, Pp 1-15 (2019) Scientific Reports |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|