Department of Chemical Engineering, University of New Hampshire, Durham, New Hampshire, USA.
Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA.
Protein Sci. 2023 Aug;32(8):e4720. doi: 10.1002/pro.4720.
Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8″ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.
磷酸二酯酶-5（PDE5）通过水解将第二信使分子 cGMP 转化为 5'-GMP，从而调节其浓度。PDE5 与勃起功能障碍和心血管疾病有关。PDE5 催化结构域中的底物结合位点被几个动态结构基序（包括α14 螺旋、M 环和 H 环）包围，这些基序已知通过目前尚未解决的结构中间体在非活性和活性构象状态之间切换。我们评估了这些结构基序在apo 状态下以及结合别构抑制剂（吴茱萸碱）或竞争性抑制剂阿伐那非时的构象动力学。我们采用增强采样的 replica exchange 溶剂标度（REST2）方法、主成分分析（PCA）、时滞独立成分分析（tICA）、分子动力学（MD）模拟和 well-tempered 元动力学模拟来探测这些结构基序中的构象变化。我们的结果支持了 PDE5 的调节机制，其中α14 螺旋在向内（低活性）构象和向外（高活性）构象之间交替，同时伴随着 H 环的α8'和α8″螺旋的折叠/展开。当别构抑制剂吴茱萸碱结合到 PDE5 时，α14 螺旋的向内（非活性）状态是首选的，从而阻止底物进入催化位点。相比之下，PDE5 的竞争性抑制剂通过占据活性位点并稳定α14 螺旋的向外构象来阻止催化。定义 PDE5 激活调节的构象动力学将有助于合理设计下一代 PDE5 活性小分子调节剂。
