Institute of Chemistry, State University of Campinas, Campinas, SP, Brazil; Institute of Physics of São Carlos, University of São Paulo, São Carlos, SP, Brazil.
Biophys J. 2013 Oct 1;105(7):1670-80. doi: 10.1016/j.bpj.2013.07.032.
Nuclear hormone receptors (NRs) are major targets for pharmaceutical development. Many experiments demonstrate that their C-terminal Helix (H12) is more flexible in the ligand-binding domains (LBDs) without ligand, this increased mobility being correlated with transcription repression and human diseases. Crystal structures have been obtained in which the H12 is extended, suggesting the possibility of large amplitude H12 motions in solution. However, these structures were interpreted as possible crystallographic artifacts, and thus the microscopic nature of H12 movements is not well known. To bridge the gap between experiments and molecular models and provide a definitive picture of H12 motions in solution, extensive molecular dynamics simulations of the peroxisome proliferator-activated receptor-γ LBD, in which the H12 was bound to a fluorescent probe, were performed. A direct comparison of the modeled anisotropy decays to time-resolved fluorescence anisotropy experiments was obtained. It is shown that the decay rates are dependent on the interactions of the probe with the surface of the protein, and display little correlation with the flexibility of the H12. Nevertheless, for the probe to interact with the surface of the LBD, the H12 must be folded over the body of the LBD. Therefore, the molecular mobility of the H12 should preserve the globularity of the LBD, so that ligand binding and dissociation occur by diffusion through the surface of a compact receptor. These results advance the comprehension of both ligand-bound and ligand-free receptor structures in solution, and also guide the interpretation of time-resolved anisotropy decays from a molecular perspective, particularly by the use of simulations.
核激素受体（NRs）是药物开发的主要靶点。许多实验表明，它们的 C 端螺旋（H12）在没有配体的情况下在配体结合域（LBD）中更加灵活，这种增加的流动性与转录抑制和人类疾病有关。已经获得了 H12 延伸的晶体结构，这表明在溶液中可能存在大振幅的 H12 运动。然而，这些结构被解释为可能的晶体学假象，因此 H12 运动的微观性质尚不清楚。为了弥合实验和分子模型之间的差距，并提供溶液中 H12 运动的明确图像，对过氧化物酶体增殖物激活受体-γ LBD 进行了广泛的分子动力学模拟，其中 H12 与荧光探针结合。对模型各向异性衰减与时间分辨荧光各向异性实验进行了直接比较。结果表明，衰减速率取决于探针与蛋白质表面的相互作用，与 H12 的灵活性相关性较小。然而，为了使探针与 LBD 的表面相互作用，H12 必须折叠在 LBD 的主体上。因此，H12 的分子流动性应该保持 LBD 的球形，以便配体结合和解离通过紧凑受体的表面扩散发生。这些结果推进了对配体结合和配体自由受体结构在溶液中的理解，也从分子角度指导了对时间分辨各向异性衰减的解释，特别是通过模拟的使用。
