Computational de novo design of a disulfide-rich miniprotein synthetic library and its application to engineer binders to neutralizing epitopes on Clostridium difficile toxins TcdA and TcdB

项目摘要 富含二硫键的迷你蛋白是一种功能强大但未被充分利用的蛋白质家族，用于试剂、诊断和治疗 申请。它们像小分子一样高度稳定，但又足够大，可以专门与蛋白质结合 具有高亲和力的靶点，因此可以很容易地用于抑制蛋白质-蛋白质相互作用。富含二硫化物 小蛋白质是自然产生的，但天然分子的工程设计具有挑战性。迫切需要…… 新技术使富含二硫键的迷你蛋白能够被设计成与任意蛋白质靶标结合， 就像抗体等其他蛋白质支架的常规做法一样。在这里，我们提出了一种计算从头设计。 使用Rosetta构建合成微型蛋白质文库的策略，该文库将普遍用于筛选蛋白质 亲和试剂。 创建蛋白质文库的典型方法是在数据库中生成大量随机序列多样性 单一蛋白质结构的局部区域。这些序列中的大多数将是不稳定的或无法结合任何 目标(例如，太极或太非极)。与其使用随机序列，我们建议显式地设计 人工合成的富含二硫化物的微蛋白文库中的每个成员。我们的设计库将包含106个不同的 显示最广泛的结合面的小蛋白。编码这个文库的基因将是 使用低聚物池合成。为了在这种规模下执行计算从头设计，我们扩展了 Rosetta中的缝合算法生成数十万个独特的微蛋白质结构和 序列。我们开发了新的过滤器来评估设计模型的质量，并量化和比较蛋白质 结构和表面。 作为这种方法的测试案例，我们将通过酵母展示来筛选我们的富含二硫键的迷你蛋白文库 对艰难梭菌肠毒素TcdA和TcdB。艰难梭菌是与医疗保健相关的主要原因 在美国感染，这两种蛋白介导其致病性。目前，唯一可用的 针对这些毒素蛋白的艰难梭菌肠道感染的治疗方法是抗体，必须注射。 且疗效不佳。同一中和表位的超稳定微型蛋白结合体可能是 口服给药。因此，这项工作为从头开始的微型蛋白质工程和文库提供了一个新的前沿 设计。它还将产生一种新的、具有治疗意义的分子，用于治疗C。 艰难梭菌感染。

Project Summary Disulfide-rich miniproteins are a powerful yet underutilized protein family for reagent, diagnostic and therapeutic applications. They are hyperstable like small molecules, yet are large enough to bind specifically to protein targets with high affinity and thus can be readily used to inhibit protein-protein interactions. Disulfide-rich miniproteins occur naturally, but the natural molecules are challenging to engineer. There is a pressing need for new technologies which enable disulfide-rich miniproteins to be engineered to bind to arbitrary protein targets, as is routine for other protein scaffolds like antibodies. Here, we propose a computational de novo design strategy using Rosetta to build a synthetic miniprotein library that will be generally useful for screening protein affinity reagents. The typical approach to creating protein libraries is to generate a large amount of random sequence diversity in a localized area of a single protein structure. Most of these sequences will be unstable or unable to bind any target (e.g. too polar or too nonpolar). Rather than use random sequences, we propose to explicitly design each member of a synthetic disulfide-rich miniprotein library. Our design library will contain 106 different miniproteins that display the widest possible variety of binding surfaces. The genes encoding this library will be synthesized using oligo pools. To perform computational de novo design at this scale, we extended the SEWING algorithm in Rosetta to generate hundreds of thousands of unique miniprotein structures and sequences. We developed novel filters to assess design model quality and to quantify and compare protein structures and surfaces. As a test case of this approach, we will screen our disulfide-rich miniprotein library via yeast display for binders to Clostridium difficile enterotoxins TcdA and TcdB. C. difficile is the leading cause of healthcare-related infections in the USA, and these two proteins mediate its pathogenicity. At present, the only available treatments for C. difficile enteric infection that target these toxin proteins are antibodies, which must be injected and have poor efficacy. A hyperstable miniprotein binder to the same neutralizing epitope could be administered orally. Therefore, this work presents a new frontier in de novo miniprotein engineering and library design. It will also result in a source of novel, therapeutically interesting molecules for the treatment of C. difficile infection.