A Sequential Binding Mechanism in a PDZ Domain
A Sequential Binding Mechanism in a PDZ Domain
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DOI：
10.1021/bi900559k
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发表时间：
2009-08-04
期刊：
影响因子：
2.9
通讯作者：
Jemth, Per
中科院分区：
文献类型：
--
作者：
Chi, Celestine N.;Bach, Anders;Jemth, Per
构象选择和诱导拟合是变构蛋白与配体相互作用的两种众所周知的机制。一些蛋白质，如泛素，最近被发现在平衡状态下以多种构象存在，这表明构象选择可能是相互作用的一般机制，特别是对于单结构域蛋白质。在这里，我们发现SAP97的PDZ2结构域通过序列（诱导配合）机制结合其配体。我们使用SAP97 PDZ2和肽配体进行了结合实验，并使用停止流动技术观察了两相动力学，表明配体结合至少涉及两步过程。通过使用超快速连续流混合器，我们检测到结合速率常数与肽浓度的双曲线依赖性，证实了两步结合机制。此外，我们还发现了PDZ和肽浓度对速率常数的类似依赖性，这表明pdz2 -肽相互作用涉及一个前络合物，然后发生构象变化，从而遵循诱导配合机制。
Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism.