Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct
The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to...
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Elsevier
2025-06-01
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| Series: | Journal of Structural Biology: X |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590152425000042 |
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| author | Suruchi Singh Yi Liu Meghan Burke Vamseedhar Rayaprolu Stephen E. Stein S. Saif Hasan |
| author_facet | Suruchi Singh Yi Liu Meghan Burke Vamseedhar Rayaprolu Stephen E. Stein S. Saif Hasan |
| author_sort | Suruchi Singh |
| collection | DOAJ |
| description | The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on N-glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination. |
| format | Article |
| id | doaj-art-4430d84dda8540978bce042a9bce5f29 |
| institution | OA Journals |
| issn | 2590-1524 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Structural Biology: X |
| spelling | doaj-art-4430d84dda8540978bce042a9bce5f292025-08-20T02:35:51ZengElsevierJournal of Structural Biology: X2590-15242025-06-011110012310.1016/j.yjsbx.2025.100123Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain constructSuruchi Singh0Yi Liu1Meghan Burke2Vamseedhar Rayaprolu3Stephen E. Stein4S. Saif Hasan5Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, USAMass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899, USAMass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899, USAPacific Northwest Cryo-EM Center, Oregon Health and Sciences University, Portland, OR 97201, USAMass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899, USADepartment of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, USA; University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore MD 21201, USA; Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Rockville MD 20850, USA; Corresponding author at: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, USA.The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on N-glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.http://www.sciencedirect.com/science/article/pii/S2590152425000042SpikeSingle particle cryoEMAlphaFoldPurification |
| spellingShingle | Suruchi Singh Yi Liu Meghan Burke Vamseedhar Rayaprolu Stephen E. Stein S. Saif Hasan Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct Journal of Structural Biology: X Spike Single particle cryoEM AlphaFold Purification |
| title | Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct |
| title_full | Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct |
| title_fullStr | Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct |
| title_full_unstemmed | Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct |
| title_short | Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct |
| title_sort | production and cryo electron microscopy structure of an internally tagged sars cov 2 spike ecto domain construct |
| topic | Spike Single particle cryoEM AlphaFold Purification |
| url | http://www.sciencedirect.com/science/article/pii/S2590152425000042 |
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