Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called “ideal” folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes.

中文翻译：

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结合抗体的计算设计原理

天然蛋白质必须折叠成稳定的构象并发挥其分子功能。迄今为止，计算设计通过使用富含规则二级结构且几乎没有环和不稳定元素（如空腔）的所谓“理想”折叠，成功地生产出稳定且原子精确的蛋白质。然而，分子功能，例如结合和催化，通常需要非理想的特征，包括大而不规则的环和掩埋的极性相互作用网络，这对于折叠设计仍然具有挑战性。通过五个设计/实验周期，我们了解了设计稳定和功能性抗体可变片段 (Fvs) 的原理。具体来说，我们 ( i ) 使用了源自抗体多序列比对的序列设计约束，并且 ( ii )) 在骨架设计期间，在天然抗体中观察到的互补决定区 (CDR) 1 和 2 的框架和环之间保持稳定的相互作用。设计的 Fv 以中纳摩尔亲和力结合它们的配体，并且与天然抗体一样稳定，尽管有 >30 个突变来自哺乳动物抗体种系。此外，晶体学分析证明了整个框架和一个设计中六个 CDR 中的四个的原子精度和另一个设计中整个 Fv 的原子精度。我们学到的原理是通用的，可以用于设计其他非理想折叠，生成稳定、特异性和精确的抗体和酶。