Dynamop Group, Institut de Biologie Structurale J.-P. Ebel, CEA CNRS UJF, UMR 5075, Grenoble cedex 01, France.
Mol Biol Evol. 2012 Jun;29(6):1683-94. doi: 10.1093/molbev/mss015. Epub 2012 Jan 19.
Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.
蛋白质以可相互转化的亚稳态动态集合体的形式存在，这决定了它们的构象能量景观。最近的研究表明，使构象亚稳态（CS）之间的平衡发生转移的突变是蛋白质进化出新功能的主要机制之一。在本研究中，我们使用来自嗜热细菌 Thermus thermophilus（Tt）的变构四聚体乳酸脱氢酶（LDH）作为模型酶，通过研究蛋白质对极端条件的表型适应来探究这一断言。在果糖 1,6 二磷酸（FBP）的存在下，变构 LDH 催化丙酮酸转化为乳酸，同时氧化还原型烟酰胺腺嘌呤二核苷酸（NADH）。催化涉及低亲和力无活性“T 态”和高亲和力活性“R 态”之间的结构转变，其中结合 FBP。在此结构转变过程中，两个重要残基的侧链构象发生变化。这些残基是 R171 和 H188，它们分别参与底物和 FBP 的结合。我们设计了两种与活性位点相距较远的 Tt-LDH 突变体（一种“1-Mut”和五种“5-Mut”），并对其催化、动力学和结构特性进行了表征。在 1-Mut Tt-LDH 中，没有 FBP 时，K(m)(Pyr)与野生型相比降低，这与 H188 的 CS 平衡完全转移到 R 态观察到的平衡一致。相比之下，R171、k(cat)和蛋白质稳定性的 CS 群体变化很小。在 5-Mut Tt-LDH 中，没有 FBP 时，K(m)(Pyr)接近有 FBP 时的值，并且几乎与温度无关，k(cat)显著增加，R171 的 CS 群体向 R 态的 CS 群体转移。这些变化伴随着在较高温度下蛋白质稳定性的降低，这与在较低温度下的柔韧性增加一致。总之，这些结果表明，仅少数甚至单个突变就可以强烈改变酶的热性质，从而改变功能重要的天然状态 CS 的平衡，进而改变它们的相对群体，而不改变 CS 本身的性质。它们还为蛋白质适应温度的突变途径类型提供了见解。
