Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
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DOI：
10.7554/elife.07574
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发表时间：
2015-09-30
期刊：
影响因子：
7.7
通讯作者：
Fraser, James S.
中科院分区：
文献类型：
--
作者：
Keedy, Daniel A.;Kenner, Lillian R.;Fraser, James S.
在原子细节上确定动态蛋白质的相互转换构象是结构生物学的一个主要挑战。动态酶亲环素A（CypA）的活性位点的构象异质性先前已被链接到其催化功能，但在何种程度上这些残基的不同构象的相关性是不清楚的。在这里，我们比较的构象合奏CypA多温同步加速器晶体学和固定靶X射线自由电子激光（XFEL）晶体学。XFEL实验的衍射破坏前的性质提供了一个辐射损伤的CypA的功能重要的替代构象的自由视图，确认早期的同步加速器为基础的结果。我们监测这些替代构象的温度依赖性与8个同步辐射数据集跨越100-310 K。多构象模型表明，CypA中的许多替代构象仅在240 K及以上填充，而其他构象在180 K及以下保持填充或变得填充。这些结果表明，180- 240 K之间的构象异质性的复杂演变，涉及热失活和溶剂驱动的蛋白质运动在晶体中的逮捕。缺乏一个单一的共享构象响应温度内的动态活性位点网络提供了证据的构象洗牌模型，其中在网络的中间的一个大的芳香环的旋转异构体状态之间的交换移动的构象合奏网络中的其他残基。总之，我们的多温度分析和XFEL数据激发了新一代的温度和时间分辨实验，以从结构上表征蛋白质功能的动态基础。
Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180--240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.