BACKGROUND This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β2-adrenergic receptor (β2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein's binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site). RESULTS In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor's activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds. CONCLUSIONS Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.

中文翻译：

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在存在细胞内环3的情况下人β2-肾上腺素能受体的变构偶联研究。

背景技术这项研究调查了在存在细胞内环3（ICL3）的情况下，人β2-肾上腺素能受体（β2-AR）的细胞内和细胞外部分之间存在的变构偶联，在所有晶体学实验和大多数到目前为止，模拟研究报告。我们最近的1μs长MD运行揭示了过渡到所谓的非常失活的受体状态，其中ICL3堆积在G蛋白的结合腔下，并完全阻止了其与G蛋白的可及性。同时，跨膜螺旋5（TM5）的向外倾斜导致细胞外配体结合位点的扩展。在目前的研究中 我们进行了总持续时间为4μs的独立运行，以进一步研究带有填充的ICL3的非常不活泼的状态和变构偶联事件（三个不受限制的运行和五个在配体结合位点具有键约束的运行）。结果在所有三个独立的不受限制的运行中（每个长度为500 ns），ICL3在研究的时间范围内保留了其最初的包装/封闭构象，表明该受体的活性受到抑制。后来在配体结合位点的一些关键残基之间施加了特定的键约束，这些残基已通过实验确定与配体相互作用。将结合位点区域限制在开放状态有助于ICL3的封闭，而相对受约束/封闭的结合位点则阻碍了ICL3的堆积。但是，相反的操作，即打开打包的ICL3，通过将结合位点区域限制在闭合状态不能实现。因此，由于存在持久的氢键，任何尝试都无法使ICL3从其锁定状态中释放出来。结论总的来说，我们的模拟表明，从非常不活跃的状态开始，受体几乎被不可逆地抑制，这反过来又降低了受体的整体迁移率。当在配体结合位点结合时，代表各种尺寸的配体所引起的几何约束的键约束会诱导TM5，TM6以及因此的ICL3发生预期的构象变化。尽管如此，一旦将ICL3装满，由于将ICL3连接到受体核心的强氢键，变构偶联仍然无效。由于存在持久的氢键，任何尝试都无法使ICL3从锁定状态中释放出来。结论总的来说，我们的模拟表明，从非常不活跃的状态开始，受体几乎被不可逆地抑制，这反过来又降低了受体的整体迁移率。当在配体结合位点结合时，代表各种尺寸的配体所引起的几何限制的键约束会诱导TM5，TM6以及因此的ICL3发生预期的构象变化。尽管如此，一旦将ICL3装满，由于将ICL3连接到受体核心的强氢键，变构偶联仍然无效。由于存在持久的氢键，任何尝试都无法使ICL3从锁定状态中释放出来。结论总的来说，我们的模拟表明，从非常不活跃的状态开始，受体几乎被不可逆地抑制，这反过来又降低了受体的整体迁移率。当在配体结合位点结合时，代表各种尺寸的配体所引起的几何限制的键约束会诱导TM5，TM6以及因此的ICL3发生预期的构象变化。尽管如此，一旦将ICL3装满，由于将ICL3连接到受体核心的强氢键，变构偶联仍然无效。受体几乎被不可逆地抑制，从而降低了受体的整体迁移率。当在配体结合位点结合时，代表各种尺寸的配体所引起的几何限制的键约束会诱导TM5，TM6以及因此的ICL3发生预期的构象变化。尽管如此，一旦将ICL3装满，由于将ICL3连接到受体核心的强氢键，变构偶联仍然无效。受体几乎被不可逆地抑制，从而降低了受体的整体迁移率。当在配体结合位点结合时，代表各种尺寸的配体所引起的几何限制的键约束会诱导TM5，TM6以及因此的ICL3发生预期的构象变化。尽管如此，一旦将ICL3装满，由于将ICL3连接到受体核心的强氢键，变构偶联仍然无效。