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

Abstract

Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We have already demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing a PA12 tag as an inserted epitope. Nevertheless, we also found that the complex formation through the inserted PA12 tag inevitably caused structural change around the insertion site of the target. Hence, we here attempted to improve the insertion method and consequently discovered that utilization of a PA14 tag significantly reduced the structural change in the target. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. Due to this structural property, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Molecular dynamics simulations also indicated that the folding of the target was rigid regardless of the PA14 insertion and the complex formation with the NZ-1 Fab. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.