Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.
Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.
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DOI：
10.1107/s2059798321002527
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发表时间：
2021-05-01
期刊：
影响因子：
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通讯作者：
Nogi T
中科院分区：
文献类型：
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作者：
Tamura-Sakaguchi R;Aruga R;Hirose M;Ekimoto T;Miyake T;Hizukuri Y;Oi R;Kaneko MK;Kato Y;Akiyama Y;Ikeguchi M;Iwasaki K;Nogi T
提出了一种使用插入的外源表位的改进的抗体标记方法，其中抗体的结合对靶标的折叠具有较小的影响。抗体标记已被广泛进行结构测定使用X射线晶体学和电子显微镜（EM）。然而，建立靶特异性抗体是应用抗体辅助结构分析的先决条件。为了扩大这种策略的适用性，已经开发了一种替代方法，通过将外源表位插入靶标来制备抗体复合物。已经证明，NZ-1单克隆抗体的Fab可以与含有PA 12标签作为插入表位的靶标形成稳定的复合物。然而，还发现通过插入的PA 12标签形成复合物不可避免地引起靶上插入位点周围的结构变化。在此，尝试改进标签插入方法，结果发现替代标签（PA 14）可以取代靶上的各种环，而不会引起大的结构变化。晶体学分析表明，插入的PA 14标签在NZ-1 Fab的抗原结合口袋中采用具有封闭末端的环状构象。由于结合构象中末端的接近，更优化的PA 14标签对靶结构仅具有微小的影响。事实上，PA 14标签也可以插入空间位阻环中进行标记。分子动力学模拟也显示了一个刚性结构的目标，无论PA 14插入和复合物的形成与NZ-1 Fab。使用这种改进的标记技术，负染EM上进行细菌的站点-2蛋白酶，这使得近似的结构域排列的基础上的对接模式的NZ-1 Fab。
An improved antibody-labeling method using an inserted exogenous epitope is presented, in which binding of the antibody has less impact on the folding of the target. 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.