Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

• Tamura-Sakaguchi, Risako

  Graduate School of Medical Life Science, Yokohama City University

• Aruga, Rie

  Graduate School of Medical Life Science, Yokohama City University

• Hirose, Mika

  Institute for Protein Research, Osaka University

• Ekimoto, Toru

  Graduate School of Medical Life Science, Yokohama City University

• Miyake, Takuya

  Institute for Frontier Life and Medical Sciences, Kyoto University

• Hizukuri, Yohei

  Institute for Frontier Life and Medical Sciences, Kyoto University

• Oi, Rika

  Graduate School of Medical Life Science, Yokohama City University

• Kaneko, Mika K.

  Department of Antibody Drug Development, Tohoku University Graduate School of Medicine

• Kato, Yukinari

  Department of Antibody Drug Development, Tohoku University Graduate School of Medicine

• Akiyama, Yoshinori

  Institute for Frontier Life and Medical Sciences, Kyoto University

• Ikeguchi, Mitsunori

  Graduate School of Medical Life Science, Yokohama City University

• Iwasaki, Kenji

  Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba

• Nogi, Terukazu

  Graduate School of Medical Life Science, Yokohama City University

Abstract

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.

Journal

• Acta Crystallographica Section D Structural Biology

  Acta Crystallographica Section D Structural Biology 77 (5), 645-662, 2021-05

  International Union of Crystallography (IUCr)

Keywords

• antibody labeling
• epitope insertion
• antibody-assisted structural analysis
• electron microscopy
• protein crystallography

Details

• CRID: 1050006507974885760
• NII Article ID: 120007032351
• DOI: 10.1107/s2059798321002527
• HANDLE: 2433/262726
• ISSN: 20597983
• Web Site: http://journals.iucr.org/d/issues/2021/05/00/ji5021/ji5021.pdf
• Text Lang: en
• Article Type: journal article
• Data Source:
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  • CiNii Articles
  • KAKEN