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Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion
Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strat...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8098476/ https://www.ncbi.nlm.nih.gov/pubmed/33950020 http://dx.doi.org/10.1107/S2059798321002527 |
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author | Tamura-Sakaguchi, Risako Aruga, Rie Hirose, Mika Ekimoto, Toru Miyake, Takuya Hizukuri, Yohei Oi, Rika Kaneko, Mika K. Kato, Yukinari Akiyama, Yoshinori Ikeguchi, Mitsunori Iwasaki, Kenji Nogi, Terukazu |
author_facet | Tamura-Sakaguchi, Risako Aruga, Rie Hirose, Mika Ekimoto, Toru Miyake, Takuya Hizukuri, Yohei Oi, Rika Kaneko, Mika K. Kato, Yukinari Akiyama, Yoshinori Ikeguchi, Mitsunori Iwasaki, Kenji Nogi, Terukazu |
author_sort | Tamura-Sakaguchi, Risako |
collection | PubMed |
description | Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab. |
format | Online Article Text |
id | pubmed-8098476 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-80984762021-05-21 Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion Tamura-Sakaguchi, Risako Aruga, Rie Hirose, Mika Ekimoto, Toru Miyake, Takuya Hizukuri, Yohei Oi, Rika Kaneko, Mika K. Kato, Yukinari Akiyama, Yoshinori Ikeguchi, Mitsunori Iwasaki, Kenji Nogi, Terukazu Acta Crystallogr D Struct Biol Research Papers Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab. International Union of Crystallography 2021-04-19 /pmc/articles/PMC8098476/ /pubmed/33950020 http://dx.doi.org/10.1107/S2059798321002527 Text en © Tamura-Sakaguchi et al. 2021 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
spellingShingle | Research Papers Tamura-Sakaguchi, Risako Aruga, Rie Hirose, Mika Ekimoto, Toru Miyake, Takuya Hizukuri, Yohei Oi, Rika Kaneko, Mika K. Kato, Yukinari Akiyama, Yoshinori Ikeguchi, Mitsunori Iwasaki, Kenji Nogi, Terukazu Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title | Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title_full | Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title_fullStr | Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title_full_unstemmed | Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title_short | Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion |
title_sort | moving toward generalizable nz-1 labeling for 3d structure determination with optimized epitope-tag insertion |
topic | Research Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8098476/ https://www.ncbi.nlm.nih.gov/pubmed/33950020 http://dx.doi.org/10.1107/S2059798321002527 |
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