Yonkunas Michael, Buddhadev Maiti, Flores Canales Jose C, Kurnikova Maria G
Chemistry Department, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Chemistry Department, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Biophys J. 2017 Jun 6;112(11):2291-2300. doi: 10.1016/j.bpj.2017.04.042.
Ionotropic glutamate receptors are a family of tetrameric ion channels with functional states consisting of nonconducting, conducting, and desensitized states that are starting to become well characterized by electrophysiological and biophysical studies. However, the structure and relative energetics of these states beyond the general structure of the receptor are still not well understood. It is known that the interface between monomeric subunits of the tetramer plays a major role in distinguishing these functional states. We have used umbrella sampling and multimicrosecond molecular dynamics simulations of the GluA2 AMPA subtype glutamate receptor ligand-binding domain (LBD) dimers to characterize a natural propensity of the LBD dimers for various configurational states. Our results show a proposed desensitized conformation of the LBD dimer is a highly preferable conformation of the LBD dimer without the influence of other receptor domains or crystallographic conditions. This has been demonstrated by both free protein simulations of 5 μs duration, as well as by computed free energy difference between the active and desensitized states. At the same time, the simulations performed using the same protocols revealed that for the LBD mutant L483Y, known to lack desensitization, the postulated active state of the LBD dimer is indeed the preferred configurational state, which remained stable in the simulations. Our findings pave the path for developing more detailed hypotheses of the full receptor activation mechanism. Combined with the energetics of glutamate binding to the LBD and the energy required to open the transmembrane pore helices, our results strongly support a hypothesis that the low absolute free-energy state is the desensitized state of the intact AMPA receptor.
离子型谷氨酸受体是一类四聚体离子通道，其功能状态包括非传导态、传导态和失敏态，电生理和生物物理研究已开始对这些状态进行充分表征。然而，除了受体的一般结构外，这些状态的结构和相对能量仍未得到很好的理解。已知四聚体的单体亚基之间的界面在区分这些功能状态中起主要作用。我们使用了GluA2 AMPA亚型谷氨酸受体配体结合结构域（LBD）二聚体的伞形采样和多微秒分子动力学模拟，以表征LBD二聚体对各种构象状态的自然倾向。我们的结果表明，在没有其他受体结构域或晶体学条件影响的情况下，LBD二聚体的一种假定失敏构象是LBD二聚体的高度优选构象。这已通过5微秒时长的游离蛋白质模拟以及活性态和失敏态之间的计算自由能差得到证明。同时，使用相同协议进行的模拟表明，对于已知缺乏失敏作用的LBD突变体L483Y，LBD二聚体的假定活性态确实是优选的构象状态，在模拟中保持稳定。我们的发现为开发更详细的完整受体激活机制假说铺平了道路。结合谷氨酸与LBD结合的能量以及打开跨膜孔螺旋所需的能量，我们的结果有力地支持了一个假说，即低绝对自由能状态是完整AMPA受体的失敏态。
