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

Risako Tamura-Sakaguchi,Yohei Hizukuri,Kenji Iwasaki,Yukinari Kato,Yoshinori Akiyama,Mika K Kaneko,Terukazu Nogi,Mika Hirose,Rika Oi,Mitsunori Ikeguchi,Rie Aruga,Takuya Miyake,Toru Ekimoto

doi:10.1107/s2059798321002527

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.

Highlights

Antibody labeling has become a useful tool for determining the structures of protein molecules and complexes
Antibody labeling has been widely used in the structural analysis of protein molecules and complexes
The use of inserted exogenous epitopes has the potential to expand the applicability of antibody labeling, even to cases where no antibodies have been established for the target protein

Summary

Introduction

Antibody labeling has become a useful tool for determining the structures of protein molecules and complexes. A large number of crystal structures have successfully been determined using antibody fragments as crystallization chaperones. Antibody labeling has been useful for determining membrane-protein structures. The first use of antibody labeling to facilitate the crystallization of a membrane protein was in the structural analysis of bacterial cytochrome c oxidase in complex with an Fv fragment (Ostermeier et al, 1995). These observations prompted the usefulness of antibody labeling has been broadly accepted the prediction that the PA12 tag could bind to NZ-1 either in structural biology. Our previous study demonstrated the utilizing antibody labeling that limits its applicability

Methods

Results

Conclusion