Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Protein Sci. 2024 Jun;33(6):e5037. doi: 10.1002/pro.5037.
The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.
噬菌体蛋白 Paratox（Prx）通过直接结合信号受体和转录因子 ComR 来阻断其链球菌宿主中的群体感应。这降低了链球菌摄取环境 DNA 的能力，并保护噬菌体 DNA 免受重组损伤。过去对 Prx：ComR 分子相互作用的研究表明，当与 ComR 结合时，Paratox 采用有序的球状折叠。然而，溶液状态的生物物理测量表明 Prx 可能具有构象动态性。为了解决这一差异，我们使用圆二色性、核磁共振和几种荧光蛋白折叠测定法研究了 Prx 在溶液中的稳定性和动态特性。我们的工作表明，在稀缓冲条件下，Prx 本质上是无定形的。我们还表明，添加高渗盐或蛋白质稳定渗透剂可诱导 Prx 折叠。然而，溶质稳定的折叠与 Prx 与 ComR 结合时采用的构象不同。此外，我们通过稳态荧光和停流动力学测量表征了 Prx 的折叠热力学和折叠动力学。我们的结果表明，Prx 在稀溶液中是一种高度动态的蛋白质，在 10 ms 的时间尺度内折叠和重新折叠。总的来说，我们的结果表明，链球菌噬菌体蛋白 Prx 在二态平衡中是一种固有无序的蛋白质，具有溶质稳定的折叠形式。此外，在溶质拥挤的细菌细胞中，溶质稳定的折叠形式可能是 Prx 的主要形式。最后，我们的工作表明，Prx 通过构象选择和诱导契合结合机制结合并抑制 ComR，从而抑制链球菌中的群体感应。
