Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia.
Venom Evolution Lab, School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.
Angew Chem Int Ed Engl. 2022 May 2;61(19):e202200951. doi: 10.1002/anie.202200951. Epub 2022 Mar 11.
Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.
Knottins 是拓扑结构复杂的肽，由半胱氨酸环稳定，具有异常多样化的功能，包括蛋白酶抑制。然而，调节其原位活性的方法有限。在这里，我们展示了使用光或链霉亲和素调节 knottin 蛋白酶抑制剂活性的两种独立方法。我们表明，通过激活第二种作用模式，可以用光来控制工程化 knottin 的抑制活性和选择性，该模式将抑制剂针对新靶标打开。通过 knottin 文库筛选，我们还确定了抑制剂结合环中的一个位置，允许插入生物素标签而不损害活性。使用链霉亲和素，具有纳摩尔亲和力的生物素化 knottin 可以在活性测定中关闭活性，并且因子 XIIa 抑制剂的抗凝活性可以在人血浆中快速关闭。我们的发现扩展了工程化 knottin 用于精确控制蛋白质功能的范围。
