Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example
Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example
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DOI：
10.1074/jbc.m110.124040
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发表时间：
2010-09-03
影响因子：
4.8
通讯作者：
Jemth, Per
中科院分区：
文献类型：
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作者：
Chi, Celestine N.;Bach, Anders;Jemth, Per
二聚体配体可以是蛋白质-蛋白质或酶-底物相互作用的有效抑制剂。由于它们同时结合两个结合位点的能力，它们对靶点的亲和力和特异性增加，因此在药物设计中具有吸引力。然而，很少有研究已经解决了这样的二价配体的相互作用的动力学机制。我们已经研究了结合最近确定的有效的血浆稳定的二聚五肽和PDZ 1 -2的突触后密度蛋白-95（PSD-95）的相互作用，使用蛋白质工程结合荧光偏振，等温滴定量热法，和停流荧光。我们证明，通过两步过程，其中一个初始的结合到两个PDZ域之一，随后是一个分子内的步骤，产生的双齿复合物的结合发生。我们已经确定了所有的速率常数参与的结合反应，并发现证据的复杂的构象转变。我们的数据证明了一个成功的二聚体配体的缓慢解离的重要性，但也突出了优化分子内缔合速率的可能性。因此，这些结果可能有助于一般的二聚体抑制剂的设计。
Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.