Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Program in Biophysics, Stanford University, Stanford, CA 94305, USA.
Mol Cell. 2022 Apr 7;82(7):1329-1342.e8. doi: 10.1016/j.molcel.2022.02.026. Epub 2022 Mar 16.
Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales.
Argonautes 是一类核酸制导的蛋白，在所有生命领域中行使多种细胞功能。目前人们对于不同进化压力如何塑造每个 Argonaute 的生物物理特性还知之甚少。我们采用高通量生物化学方法来表征真细菌 Argonaute（TtAgo），一种 DNA 制导的 DNA 内切酶，如何找到、结合和切割其靶标。我们发现 TtAgo 利用与真核 Argonautes 相似的生物物理适应性来实现快速结合，但需要更广泛的互补性才能实现高亲和力的靶标结合。利用这些数据，我们构建了 TtAgo 结合速率和平衡结合亲和力的模型，估算了靶相互作用能量的核酸和蛋白质介导成分。最后，我们表明 TtAgo 的切割速率因 DNA 向导而异，这表明只有一小部分向导能够在生理相关的时间尺度上切割靶标。
