Interconversion of Functional Motions between Mesophilic and Thermophilic Adenylate Kinases

Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō) simulation of mesophilic AK from E. coli (AKmeso) to simulations of thermophilic AK from Aquifex aeolicus (AKthermo). In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O) ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS) ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A “heated” simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the “corresponding states” hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.动态特性在众多蛋白质中具有关键的功能意义，以腺苷酸激酶（adenylate kinase, AK）为例，其开/关构象转变直接限制了催化周转速率。本研究将我们此前发表的大肠杆菌嗜温型腺苷酸激酶（AKmeso）的粗粒度（双势阱Gō）模拟结果，与水生栖热菌嗜热型腺苷酸激酶（AKthermo）的模拟结果进行对比。在AKthermo中，与AKmeso一致，配体存在时LID结构域优先于核苷单磷酸（nucleoside monophosphate, NMP）结构域发生闭合，但AKthermo开放态（O）集合中的LID刚体柔性显著降低。相较于AKmeso，AKthermo的开放态和/或过渡态（TS）集合中，部分域间主链铰链区以及LID结构域内的主链折叠程度显著提升。在接触空间层面，AKthermo的过渡态在核心（CORE）结构域-LID结构域界面的接触数更少，但在核心（CORE）结构域-核苷单磷酸（NMP）结构域界面周围形成了更强的接触网络。在375K下开展的高温模拟结果显示，AKthermo的LID刚体柔性略有提升，这与对应态假说相符。此外，尽管将AKthermo中的7个脯氨酸（proline）位点虚拟突变为AKmeso的对应残基，仅引发了相似的微小扰动，但要使LID刚体柔性与铰链柔性达到AKmeso的水平，需将这些位点（尤其是第8位和第155位）突变为甘氨酸（glycine）。将AKmeso中的这7个位点突变为脯氨酸（proline）后，部分铰链区的柔性有所降低（尤其是铰链2），但并未削弱LID的刚体柔性，这表明AKmeso中的这两类运动是解耦的。综上，本研究结果表明，铰链柔性与全局功能运动既存在相关性，又并非完全由铰链残基所决定。该突变框架可为其他蛋白质中具有功能重要性的柔性与别构效应（allostery）的理性设计提供参考，助力新型生化通路的工程化构建。