Centre National de la Recherche Scientifique (CNRS)
France
Other private
Citation
Journal: J Struct Biol / Year: 2024 Title: Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex. Authors: Rémi Ruedas / Rémi Vuillemot / Thibault Tubiana / Jean-Marie Winter / Laura Pieri / Ana-Andreea Arteni / Camille Samson / Slavica Jonic / Magali Mathieu / Stéphane Bressanelli / Abstract: Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody- ...Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.
Entire : Ternary complex of her2-trastuzumab-pertuzumab Fabs
Entire
Name: Ternary complex of her2-trastuzumab-pertuzumab Fabs
Components
Complex: Ternary complex of her2-trastuzumab-pertuzumab Fabs
Protein or peptide: Trastuzumab fab light chain
Protein or peptide: Trastuzumab Fab heavy chain
Protein or peptide: Pertuzumab Fab light chain
Protein or peptide: Pertuzumab Fab heavy chain
Protein or peptide: Receptor tyrosine-protein kinase erbB-2
Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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Supramolecule #1: Ternary complex of her2-trastuzumab-pertuzumab Fabs
Supramolecule
Name: Ternary complex of her2-trastuzumab-pertuzumab Fabs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 Details: Fab fragment from trastuzumab and pertuzumab linked to the extracellular domain of erbb2 (her2)
Source (natural)
Organism: Homo sapiens (human)
Molecular weight
Theoretical: 171.364 KDa
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Macromolecule #1: Trastuzumab fab light chain
Macromolecule
Name: Trastuzumab fab light chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288.15 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
TFS GLACIOS
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 8928 / Average exposure time: 4.71 sec. / Average electron dose: 60.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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