Falbo Emanuele, Delre Pietro, Lavecchia Antonio
Department of Pharmacy, "Drug Discovery Laboratory", University of Naples Federico II, via Domenico Montesano 49, I-80131 Naples, Italy.
ACS Omega. 2025 Feb 17;10(13):13303-13318. doi: 10.1021/acsomega.4c11128. eCollection 2025 Apr 8.
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor whose ligand-induced conformational changes, primarily driven by helix 12 (H12) repositioning, regulate transcriptional activity. However, the precise mechanism remains elusive. In this study, we performed classical molecular dynamics (cMD) simulations of the PPARγ ligand binding domain (LBD) in complex with two agonists (BRL, 3EA), a partial agonist (GW0072), and an antagonist (EKP), generating 3 μs trajectories for each system. To gain deeper insights, we integrated machine learning-assisted clustering with MD simulations, revealing a favorable trend in binding free energy (Δ ), suggesting enhanced complex stability. A case study on EKP demonstrated that, despite fitting within the binding site, it failed to induce rapid LBD or H12 rearrangements in the apo agonist-induced conformation. Additionally, we investigated the apo-state conformations of PPARγ-LBD influenced by agonist and antagonist ligands, utilizing cMD and Gaussian accelerated molecular dynamics (GaMD) over a cumulative 6 μs (3 μs cMD + 3 μs GaMD). Key residues known to modulate PPARγ function upon mutation were analyzed, and simulations confirmed the high stability of both apo and ligand-bound conformations. Notably, in the apo state, specific H12 residues interacted with other PPARγ-LBD regions, preventing disorder and abrupt transitions. These findings guided the selection of collective variables (CVs) for well-tempered metadynamics (WT-MetaD) simulations, which-in the apo-agonist state-captured the H12 shift from agonist- to antagonist-like conformations, consistent with resolved X-ray structures. Overall, this computational framework provides novel insights into PPARγ-LBD conformational dynamics and establishes a valuable approach for rationally assessing the effects of modulators on PPARγ activity.
过氧化物酶体增殖物激活受体γ（PPARγ）是一种核受体，其配体诱导的构象变化主要由螺旋12（H12）重新定位驱动，从而调节转录活性。然而，确切机制仍不清楚。在本研究中，我们对PPARγ配体结合域（LBD）与两种激动剂（BRL、3EA）、一种部分激动剂（GW0072）和一种拮抗剂（EKP）形成的复合物进行了经典分子动力学（cMD）模拟，每个系统生成了3微秒的轨迹。为了获得更深入的见解，我们将机器学习辅助聚类与分子动力学模拟相结合，揭示了结合自由能（Δ）的有利趋势，表明复合物稳定性增强。对EKP的案例研究表明，尽管它适合结合位点，但在无配体激动剂诱导的构象中，它未能诱导LBD或H12快速重排。此外，我们利用cMD和高斯加速分子动力学（GaMD），在累计6微秒（3微秒cMD + 3微秒GaMD）的时间内研究了激动剂和拮抗剂配体影响下的PPARγ-LBD无配体状态构象。分析了已知在突变时调节PPARγ功能的关键残基，模拟证实了无配体和配体结合构象的高稳定性。值得注意的是，在无配体状态下特定的H12残基与PPARγ-LBD的其他区域相互作用，防止无序和突然转变。这些发现指导了用于温度自适应元动力学（WT-MetaD）模拟的集体变量（CVs）的选择，在无配体激动剂状态下，该模拟捕捉到了H12从激动剂样构象向拮抗剂样构象的转变，与解析的X射线结构一致。总体而言，这个计算框架为PPARγ-LBD构象动力学提供了新的见解，并建立了一种合理评估调节剂对PPARγ活性影响的有价值方法。
