Physics Department and Center of Functional Protein Assemblies, Technical University of Munich, 85748 Garching, Germany.
Physics Department and Center of Functional Protein Assemblies, Technical University of Munich, 85748 Garching, Germany.
Biophys J. 2022 Jun 21;121(12):2330-2344. doi: 10.1016/j.bpj.2022.05.023. Epub 2022 May 20.
Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid β (Aβ) peptides of different lengths. Longer Aβ peptides are associated with the formation of neurotoxic plaques related to Alzheimer's disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aβ fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.
淀粉样前体蛋白（APP）被 γ-分泌酶依次切割和加工，生成不同长度的淀粉样 β（Aβ）肽。较长的 Aβ 肽与阿尔茨海默病相关的神经毒性斑块的形成有关。基于 γ-分泌酶与 APP 底物结合的结构，我们使用分子动力学模拟研究了酶-底物相互作用，并生成了代表加工反应中依次切割中间产物的模型结构。模拟表明存在一个内部对接位点，提供了强大的酶-底物包装相互作用。在酶-底物复合物中，它位于靠近底物螺旋构象中断的区域，并沿延伸构象向活性位点继续延伸。内部对接位点由两个非极性口袋组成，优先填充大的疏水性或芳香性底物侧链以稳定结合。较小的残基（如甘氨酸）的放置会在模拟过程中引发切割模式的转变，或导致底物结合不稳定。较小残基的减少包装也会影响活性位点的水合作用和催化活性状态的形成。对加工底物中间产物和底物 G33I 突变的模拟为该突变导致短 Aβ 片段产量相对增加的实验观察结果提供了解释。此外，对底物 K28A 突变的研究表明，内部对接位点由于在加工过程中 K28 引起的膜边界处的疏水性不匹配以及底物向酶活性位点的移动，与底物解离的趋势相反。所提出的内部对接位点也可能有助于新的 γ-分泌酶调节剂的特异性设计。
