Computational design of potent and selective BAK and BAX binders

There are numerous binders of the pro-survival BCL2 family proteins such as BCL2, MCL1, and BCL-XL, but development of potent and selective binders of their pro-apoptotic counterparts BAK and BAX has remained a major unsolved challenge. We use computational protein design to generate 13 kDa binders of BAK and BAX with 400 pM and 3 nM affinity, orders of magnitude higher than any existing native or designed binder, and with greater than 100-fold specificity against pro-survival BCL2 family members. The crystal structure of the BAKᐧɑBAK2 complex is very close to the computational design model, with the binder making specific interactions extending out from the canonical BH3-binding groove. Liposome- and cell-based analyses reveal that ɑBAK2 inhibits membrane permeabilization when in excess of BAK, but activates BAK when BAK is in excess. Structural analyses indicate that binding of ɑBAK2 results in partial unfolding and exposure of BAK’s BH3 domain. Similar to ɑBAK2, ɑBAX2 activates BAX at low concentrations and does not activate BAX at high concentrations. This work provides valuable insight into design of small molecule or protein inhibitors of BAK and BAX; inhibition requires high affinity binding as well as a saturating concentration of binder at the site of action. Our designs are the first binders with the high specificity required for efficient modulation of apoptosis via direct interaction with BAK and BAX and they provide highly selective molecular probes for addressing outstanding cell biological questions about cell death.目前已开发出多种靶向抗凋亡B细胞淋巴瘤因子2（BCL2）家族蛋白的结合剂，包括B细胞淋巴瘤因子2、髓系细胞白血病1（MCL1）及BCL2样蛋白X（BCL-XL）等家族成员，但开发针对其促凋亡同源蛋白BAK与BAX的强效、选择性结合剂，仍是一项尚未解决的重大挑战。本研究借助计算蛋白质设计技术，成功获得分子量为13 kDa的BAK与BAX靶向结合剂，对BAK的亲和力达400 pM，对BAX的亲和力达3 nM，其结合活性较现有天然或人工设计的结合剂高出数个数量级，且针对抗凋亡BCL2家族蛋白的选择性超过100倍。BAK·ɑBAK2复合物的晶体结构与计算设计模型高度吻合，该结合剂通过特异性相互作用，从经典BH3结合凹槽向外延伸形成专属作用界面。脂质体实验与细胞实验分析结果表明，当ɑBAK2浓度高于BAK时，该结合剂可抑制细胞膜通透化；而当BAK浓度占优时，ɑBAK2则会激活BAK。结构分析显示，ɑBAK2与BAK的结合会导致BAK的BH3结构域部分解折叠并暴露。与ɑBAK2类似，ɑBAX2在低浓度下可激活BAX，高浓度下则不会激活BAX。本研究为BAK与BAX的小分子及蛋白质抑制剂设计提供了重要的理论参考：若要实现对这两种蛋白的有效抑制，结合剂需具备高亲和力，且在作用位点达到饱和浓度。本研究设计的结合剂是首款可通过直接靶向BAK与BAX，实现细胞凋亡有效调控所需高特异性的结合剂，同时也为解决细胞死亡领域尚存的细胞生物学难题提供了高选择性的分子探针。