Ma Dejian, Brandon Nicole R, Cui Tanxing, Bondarenko Vasyl, Canlas Christian, Johansson Jonas S, Tang Pei, Xu Yan
Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15260, USA.
Biophys J. 2008 Jun;94(11):4454-63. doi: 10.1529/biophysj.107.117838. Epub 2008 Feb 29.
The four-alpha-helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/L38M)(2,) with designed specific binding pockets for volatile anesthetics. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. The two helices from the N-terminus to the linker (helices 1 and 1') are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2') are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The site of the L38M mutation, which was previously shown to increase the halothane binding affinity by approximately 3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R(2)) rate. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia.
四螺旋束模拟了半胱氨酸环受体家族的跨膜结构域，该结构域被认为是全身麻醉药的蛋白质靶点。我们利用高分辨率核磁共振技术解析了一个典型的二聚体四螺旋束（Aalpha(2)-L1M/L38M）(2)的结构（蛋白质数据库编号：2I7U），该四螺旋束具有为挥发性麻醉药设计的特定结合口袋。螺旋-转角-螺旋基序的两个单体按最初设计形成反平行二聚体，但高分辨率结构显示四个螺旋呈不对称四级排列。从N端到连接区的两个螺旋（螺旋1和1'）在二聚体中通过F12和W15沿长疏水核心的侧链环堆积以及L4、L11、L18和L25互补对之间近乎完美的疏水相互作用延伸相互关联，所有这些氨基酸都位于沿螺旋-螺旋二聚体界面的七肽e位。相比之下，从连接区到C端的两个螺旋（螺旋2和2'）的轴彼此相距更远，在K47周围形成了一条从水相到设计的疏水核心中心的侧向通道。先前显示L38M突变位点可使氟烷结合亲和力提高约3.5倍，它并非推测参与麻醉药结合的疏水核心的一部分，但显示横向弛豫（R(2)）速率升高。通过降低谱密度映射对蛋白质动力学进行定性分析发现，螺旋中许多残基的弛豫存在交换贡献。使用无模型方法的定量分析以及核磁共振弛豫色散测量证实了这一观察结果。核磁共振结构和自动对接分析表明，对于麻醉药结合具有最有利两亲性的口袋位于二聚体疏水核心中心的W15侧链之间，每个单体的F12和F52环堆积之间可能还有另外两个较小的结合位点。设计的麻醉药结合蛋白的高分辨率结构提供了关于麻醉药-蛋白质相互作用可能位点的前所未有的原子细节，这对于理解全身麻醉的分子机制至关重要。
