Application of the NZ-1 Fab as a crystallization chaperone for PA tag-inserted target proteins

Risako Tamura, Rika Oi, Satoko Akashi, Mika K. Kaneko, Yukinari Kato, Terukazu Nogi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

Original languageEnglish
Pages (from-to)823-836
Number of pages14
JournalProtein Science
Volume28
Issue number4
DOIs
Publication statusPublished - 2019 Apr

Keywords

  • antigen-binding fragment
  • crystallization chaperone
  • epitope tag insertion
  • monoclonal antibody
  • protein crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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