CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, PSL University, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005, Paris, France.
Phys Chem Chem Phys. 2020 Sep 7;22(33):18361-18373. doi: 10.1039/d0cp02738c. Epub 2020 Aug 13.
A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.
生命在地球上的进化的一个关键方面是蛋白质适应在非常宽的温度条件下稳定和工作。对相关分子机制的详细了解也有助于设计用于生物技术过程的优化酶。尽管取得了重要进展，但对于嗜热酶如何在极端温度下成功发挥作用的综合认识仍然不完整。在这里，我们研究了中温单体大肠杆菌（Ec）和嗜热二聚体海洋栖热菌（Tm）同系物的稳定性和灵活性对温度的依赖性。我们使用全原子分子动力学模拟和复制交换方案，该方案允许增强构象采样，同时提供对酶在升温时行为的详细了解。我们表明，这种方法再现了两个同源物之间的稳定性变化，并通过识别随着温度升高而熔化的二级结构元素的序列来提供变性机制的分子描述，这在实验中不容易获得。通过在假设的 TmDHFR 单体上重复我们的方法，我们进一步确定了序列和寡聚化在 TmDFHR 异常稳定性中的各自作用。我们表明，蛋白质柔韧性和热稳定性相关的直观期望并不成立。最后，我们的模拟表明，在远低于熔点的温度下就已经发生了显著的构象波动。虽然 TmDHFR 和 EcDHFR 催化活性之间的差异通常通过涉及关键 M20 环的开放和闭合构象的简化两态图进行解释，但我们的模拟表明，两个同源物的显著不同的活性温度依赖性是由配体-辅因子距离分布的变化引起的，以响应这些构象变化。
