Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα.
Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα.
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探讨蛋白激酶 Cα C1B 结构域中二酰甘油结合亲和力的决定因素。
DOI：
10.1016/j.jmb.2011.03.020
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发表时间：
2011
影响因子：
5.6
通讯作者：
Igumenova,TatyanaI
中科院分区：
文献类型：
--
作者：
Stewart,MikaelaD;Morgan,Brittany;Massi,Francesca;Igumenova,TatyanaI
C1 结构域是独立折叠的模块，负责将其母体蛋白靶向含有二酰基甘油 (DAG)（一种普遍存在的第二信使）的脂膜。 C1 结构域的 DAG 结合亲和力决定了信号响应传播所需的 DAG 阈值浓度以及细胞中 DAG 受体之间此响应的选择性。目前可用于 C1 结构域的结构信息几乎无法深入了解其差异 DAG 结合亲和力的分子基础。在这项工作中，我们使用溶液 NMR 方法和分子动力学模拟来表征蛋白激酶 Cα (C1Bα) 的 C1B 结构域及其诊断突变体 Y123W。该突变不会扰乱 C1Bα 结构或蛋白质主链的亚纳秒动力学，但导致 DAG 结合亲和力增加 100 倍以上，并导致微秒时间尺度构象动力学发生显着变化（通过 NMR 旋转框架弛豫色散方法进行量化）。野生型和 Y123W C1Bα 之间构象交换行为的差异位于配体结合环的铰链区。分子动力学模拟提供了对交换构象异构体身份的深入了解，并揭示了特定残基 (Gln128) 在调节配体结合位点几何形状中的重要性。结合结合研究的结果，我们的研究结果表明，色氨酸侧链的构象动力学和优先分配到水-脂质界面中是调节 C1 结构域的 DAG 结合特性的重要因素。
C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.