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首页> 外文期刊>Acta crystallographica. Section D, Structural biology >Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion
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Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

机译：朝着可概括的NZ-1标签对3 d结构测定与优化epitope-tag插入

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摘要

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.
机译：抗体标记已广泛开展使用x射线结构测定晶体学和电子显微镜(EM)。然而,建立有针对性的抗体是应用antibody-assisted的先决条件结构分析。这种策略的替代方法准备开发一种抗体复杂插入一个外源抗原决定基到目标。它已被证明的工厂NZ-1单克隆抗体可以形成一个稳定的复杂的包含PA12标签作为一个目标插入表位。通过插入PA12复杂地层标签不可避免地导致结构变化插入网站目标。尝试提高就可以了方法,因此发现另一个标签(PA14)可以取代各种循环在目标没有诱导大结构性的变化。证明了插入PA14标签采用环构象与封闭的结束antigen-binding NZ-1工厂的口袋里。接近终点的只有构象,最佳PA14标签未成年人对目标结构的影响。PA14标记也可以插入位阻循环标签。分子动力学模拟也显示不论PA14刚性结构为目标与NZ-1插入和复杂的形成工厂。negative-stain EM进行细菌site 2蛋白酶,使一个近似域的安排根据对接NZ-1工厂的模式。

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