Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
Department of Biology, ETH Zurich, 8093 Zurich, Switzerland.
Nucleic Acids Res. 2024 Sep 23;52(17):10683-10704. doi: 10.1093/nar/gkae713.
The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.
多嘧啶序列结合蛋白（PTB）的 N 端 RNA 识别模体域（RRM1）形成额外的 C 端α3 螺旋，该螺旋与包含 UCUUU 五核苷酸环的茎环 RNA 结合后，停靠在β-折叠的一侧。重要的是，α3 不与 RNA 接触。因此，α3 螺旋代表一种变构方式，可以在结合结构 RNA 时调节 PTB 中相邻结构域的构象。在这里，我们使用 NMR 和 MD 研究了茎环 RNA 和 RRM1 的动态适应过程，以获得关于这种变构如何实现的机制见解。自由蛋白的弛豫数据和 NMR 结构测定表明，α3 部分有序，并与结构域短暂相互作用。茎环 RNA 结合使快速时间尺度动力学猝灭，α3 变得有序，但在蛋白-RNA 界面上观察到微秒动力学。MD 显示了 RRM1 与茎环 RNA 的结合如何与 C 端螺旋的稳定相关联，并有助于将 RNA 环序列中的差异转化为α3 长度和有序性的变化。全长 PTB 和在α3 区域具有改变动力学的突变体的 IRES 测定表明，这种动态变构影响了培养的 HEK293T 细胞中 PTB 的功能。
