ATP13A2 is a gene encoding a protein of the P5B subfamily of ATPases and is a PARK gene. Molecular defects of the gene are mainly associated with variations of Parkinson's Disease (PD). Despite the established importance of the protein in regulating neuronal integrity, the three-dimensional structure of the protein currently remains unresolved crystallographically. We have modelled the structure and reactivity of the full-length protein in its E1-ATP state. Using Molecular Dynamics (MD), Quantum cluster and Quantum Mechanical/Molecular mechanical (QM/MM) methods, we aimed at describing the main catalytic reaction, leading to the phosphorylation of Asp513. Our MD simulations suggest that two positively charged Mg2+ cations are present at the active site during the catalytic reaction, stabilizing a specific triphosphate binding mode. Using QM/MM calculations, we subsequently calculated the reaction profiles for the phosphoryl transfer step in the presence of one and two Mg2+ cations. The calculated barrier heights in both cases are found to be ~12.5 and 7.0 kcal mol-1, respectively. We elucidated details of the catalytically competent ATP conformation and the binding mode of the second Mg2+ cofactor. We also examined the role of the conserved Arg686 and Lys859 catalytic residues. We observed that by lowering significantly the barrier height of the ATP cleavage reaction, Arg686 had significant effect on the reaction. The removal of Arg686 increased the barrier height for the ATP cleavage by more than 5.0 kcal mol-1 while the removal of key electrostatic interactions created by Lys859 to the gamma-phosphate and Asp513 destabilizes the reactant state. When missense mutations occur in close proximity to an active site residue, they can interfere with the barrier height of the reaction, which can halt the normal enzymatic rate of the protein. We also found large binding pockets in the full-length structure, including a transmembrane domain pocket, which is likely where ATP13A2 cargo binds.

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来自模拟的 ATP13A2 (PARK9) 的结构动力学和催化机制

ATP13A2是编码ATP酶P5B亚家族蛋白质的基因，是PARK基因。该基因的分子缺陷主要与帕金森病 (PD) 的变异有关。尽管蛋白质在调节神经元完整性方面的重要性已确立，但蛋白质的三维结构目前仍未通过晶体学进行解析。我们对处于 E1-ATP 状态的全长蛋白质的结构和反应性进行了建模。使用分子动力学 (MD)、量子簇和量子力学/分子力学 (QM/MM) 方法，我们旨在描述导致 Asp513 磷酸化的主要催化反应。我们的 MD 模拟表明，在催化反应过程中，活性位点存在两个带正电荷的 Mg2+ 阳离子，稳定了特定的三磷酸盐结合模式。使用 QM/MM 计算，我们随后计算了在一个和两个 Mg2+ 阳离子存在下磷酰基转移步骤的反应曲线。两种情况下计算的势垒高度分别为~12.5 和 7.0 kcal mol-1。我们阐明了具有催化能力的 ATP 构象和第二个 Mg2+ 辅因子的结合模式的细节。我们还检查了保守的 Arg686 和 Lys859 催化残基的作用。我们观察到，通过显着降低 ATP 裂解反应的势垒高度，Arg686 对反应具有显着影响。Arg686 的去除使 ATP 裂解的势垒高度增加了 5.0 kcal mol-1 以上，而 Lys859 与 γ-磷酸盐和 Asp513 产生的关键静电相互作用的去除使反应物状态不稳定。当错义突变发生在靠近活性位点残基的位置时，它们会干扰反应的屏障高度，从而阻止蛋白质的正常酶促速率。我们还在全长结构中发现了大的结合口袋，包括一个跨膜域口袋，这可能是 ATP13A2 货物结合的地方。