Department of Chemistry and Biochemistry, Ohio University, Biochemistry Research Facility 108, 350 W. State St., Athens, OH, 45701, USA.
Molecular and Cellular Biology Program, Ohio University, Athens, OH, 45701, USA.
Protein J. 2018 Oct;37(5):428-443. doi: 10.1007/s10930-018-9791-9.
The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of "druggable" protein-protein interactions.
B 细胞淋巴瘤 2 (BCL2) 蛋白是一组进化上相关的蛋白，它们作为内在凋亡途径的正或负调节剂发挥作用。细胞中抗凋亡 BCL2 蛋白的过表达与凋亡抵抗有关，这可能导致癌变表型和致病细胞存活。因此，抗凋亡 BCL2 蛋白作为治疗靶点引起了相当大的关注。我们最近报道了一种基于海兔毒素 (ScTx) 的新型合成蛋白的开发，该蛋白旨在模拟促凋亡 BCL2 蛋白 Bax 的螺旋 BH3 相互作用结构域。这些研究表明，ScTx-Bax 结构中天然二硫键的数量和位置显著影响这些构建体在体外靶向抗凋亡 BCL2 蛋白的能力。本研究的目的是研究两个二硫键在 ScTx-Bax 蛋白折叠和生物学活性中的作用。在这里，我们报告了三个 ScTx-Bax 序列变体的全化学合成，每个变体在折叠结构内的不同位置都有两个天然二硫键。结果表明，两个二硫键足以使 ScTx-Bax 蛋白折叠成类似于野生型 ScTx 的天然样结构。此外，我们表明，选择的（双）二硫键 ScTx-Bax 变体可以在体外靶向 Bcl-2（适当），并且二硫键的位置显著影响结合亲和力。尽管与 Bcl-2 的结合仅略有亲和力，但含有两个二硫键的 ScTx-Bax 蛋白的成功合成代表了一种可行的途径，可以获得保留天然样构象的基于 ScTx 的 BH3 结构域模拟物。最后，ScTx-Bax 蛋白与 Bcl-2 复合物的结构模型表明，这些螺旋模拟物以与野生型 Bax BH3 结构域相似的构象结合。总之，这些结果表明 ScTx-Bax 蛋白可能作为有效的先导化合物，扩展“可成药”蛋白-蛋白相互作用的范围。
