Schmid Nathan, Zagrovic Bojan, van Gunsteren Wilfred F
Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH, CH-8093 Zürich, Switzerland.
Biochemistry. 2007 Jun 5;46(22):6500-12. doi: 10.1021/bi6026133. Epub 2007 May 12.
The polypyrimidine tract binding protein (PTB) is involved in many physiological processes, including alternative splicing, internal ribosomal entry side (IRES)-mediated initiation of translation, and polyadenylation, as well as in ensuring mRNA stability. However, the role of PTB in these processes is not fully understood, and this has motivated us to undertake a computational study of the protein. PTB RNA binding domains (RBDs) 3 and 4 and their complexes with oligopyrimidine RNAs were simulated using the GROMOS simulation software using the GROMOS 45A4 force field. First, the stability and fluctuations of the tertiary fold and of the secondary structural elements in individual domains, the combined RBD34 domain, and their complexes with RNA were studied. Second, the simulation results were validated against the experimental NMR NOE data. The analysis of hydrogen bonding patterns, salt bridge networks, and stacking interactions of the RNA to the binding pockets of the protein domains showed that binding is not sequence-specific and that many RNA fragments can bind to them successfully. Further calculations of the relative free energy of binding for different polypyrimidine sequences were carried out using the thermodynamic integration (TI) and single-step perturbation (SSP) methods. It is was not possible to calculate the relative free energies with high accuracy, but the obtained results do give qualitative insights into PTB's affinity for different RNA sequences. Furthermore, the low-energy conformations of the complexes that were found provided additional information about the mechanism of binding.
多嘧啶序列结合蛋白（PTB）参与许多生理过程，包括可变剪接、内部核糖体进入位点（IRES）介导的翻译起始和多聚腺苷酸化，以及确保mRNA的稳定性。然而，PTB在这些过程中的作用尚未完全了解，这促使我们对该蛋白进行计算研究。使用GROMOS 45A4力场，利用GROMOS模拟软件对PTB的RNA结合结构域（RBD）3和4及其与寡嘧啶RNA的复合物进行了模拟。首先，研究了单个结构域、组合的RBD34结构域及其与RNA复合物的三级结构折叠以及二级结构元件的稳定性和波动情况。其次，根据实验NMR NOE数据对模拟结果进行了验证。对RNA与蛋白质结构域结合口袋之间的氢键模式、盐桥网络和堆积相互作用的分析表明，结合不具有序列特异性，许多RNA片段都能成功与之结合。使用热力学积分（TI）和单步微扰（SSP）方法对不同多嘧啶序列的相对结合自由能进行了进一步计算。虽然无法高精度计算相对自由能，但所得结果确实为PTB对不同RNA序列的亲和力提供了定性见解。此外，所发现的复合物低能构象为结合机制提供了额外信息。
