Lasso peptides are a diverse class of naturally occurring, highly stable molecules kinetically trapped in a distinctive [1]rotaxane conformation. How the ATP-dependent lasso cyclase constrains a relatively unstructured substrate peptide into a low entropy product has remained a mystery owing to poor enzyme stability and activity in vitro. In this study, we combined substrate tolerance data with structural predictions, bioinformatic analysis, molecular dynamics simulations and mutational scanning to construct a model for the three-dimensional orientation of the substrate peptide in the lasso cyclase active site. Predicted peptide cyclase molecular contacts were validated by rationally engineering multiple, phylogenetically diverse lasso cyclases to accept substrates rejected by the wild-type enzymes. Finally, we demonstrate the utility of lasso cyclase engineering by robustly producing previously inaccessible variants that tightly bind to integrin αvβ8, which is a primary activator of transforming growth factor β and, thus, an important anti-cancer target.

套索肽是一类多种天然存在的、高度稳定的分子，动力学捕获在独特的 [1] 轮烷构象中。由于酶在体外的稳定性和活性较差，ATP 依赖性套索环化酶如何将相对非结构化的底物肽抑制成低熵产物，这仍然是一个谜。在这项研究中，我们将底物耐受性数据与结构预测、生物信息学分析、分子动力学模拟和突变扫描相结合，构建了套索环化酶活性位点底物肽的三维取向模型。通过合理设计多个系统发育多样化的套索环化酶来接受被野生型酶排斥的底物，验证了预测的肽环化酶分子接触。最后，我们通过稳健地产生以前无法接近的变体来证明套索环化酶工程的实用性，这些变体与整合素 αvβ8 紧密结合，整合素 αvβ8 是转化生长因子β的主要激活剂，因此是一个重要的抗癌靶点。