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分泌酶依次裂解和加工，产生不同长度的淀粉样β (A β ) 肽。较长的 A β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-分泌酶的APP底物结合结构，我们利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过程中顺序裂解的中间体的模型结构。模拟表明内部对接位点提供了强的酶-底物堆积相互作用。在酶-底物复合物中，它位于底物螺旋构象被中断的区域附近，并以延伸构象继续向活性位点延伸。内部对接位点由两个非极性口袋组成，这些口袋优先由大的疏水或芳香底物侧链填充以稳定结合。较小残基（例如甘氨酸）的放置可能会在模拟过程中引发裂解模式的变化，或导致底物结合不稳定。较小残基减少的堆积也会影响活性位点的水合和催化活性状态的形成。对加工后的底物中间体和底物 G33I 突变的模拟为实验观察到的该突变的短 A β片段产量的相对增加提供了解释。 此外，对底物 K28A 突变的研究表明，在加工和底物向酶活性位点移动过程中，由于 K28 引起的膜边界疏水性不匹配，内部对接位点阻止了底物解离的趋势。所提出的内部对接位点也可用于新的γ-分泌酶调节剂的具体设计。