Girard Eric, Lopes Pedro, Spoerner Michael, Dhaussy Anne-Claire, Prangé Thierry, Kalbitzer Hans Robert, Colloc'h Nathalie
Univ. Grenoble Alpes, CEA, CNRS, IBS Grenoble France.
Institute of Biophysics and Physical Biochemistry, Centre of Magnetic Resonance in Chemistry and Biomedicine, University of Regensburg Regensburg Germany
Chem Sci. 2022 Jan 13;13(7):2001-2010. doi: 10.1039/d1sc05488k. eCollection 2022 Feb 16.
In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg·GppNHp and Ras(D33K)·Mg·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.
在这项工作中，我们使用高压晶体学对Ras癌基因蛋白构象状态之间的变构转变进行了实验研究。Ras蛋白是一种小GTP酶，参与多种构象状态下发生的核心调节过程。Ras作为活性GTP结合态和非活性GDP结合态之间的分子开关，控制着重要的信号转导途径。效应器结合区域和膜相互作用区域之间的变构相互作用网络参与了Ras循环。在溶液中同时共存的构象状态比基态具有更高的吉布斯自由能。这些状态之间的平衡可以通过施加压力来改变，压力有利于具有较低偏摩尔体积的构象，并且此前已通过高压核磁共振光谱进行了分析。高压大分子晶体学（HPMX）是一种与高压核磁共振完美互补的强大工具，能够在分子水平上高分辨率地表征Ras循环中涉及的不同变构状态。我们观察到晶体在300 MPa以上发生转变，导致形成更稳定的构象体。因此，我们比较了不同高静水压力下Ras（wt）·Mg·GppNHp和Ras（D33K）·Mg·GppNHp的晶体结构。这有助于深入了解构象体之间变构平衡中涉及的结构可塑性的每个残基描述。我们已经在原子分辨率上绘制出了Ras蛋白的不同片段，这些片段保持基态构象或经历结构变化，采用与瞬态中间态相对应的激发能构象。这种由压力诱导的相变为精细探索与Ras变构亚态之间转换相关的结构决定因素提供了可能性，而无需任何突变或外源伴侣。
