Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
J Mol Biol. 2012 Mar 30;417(3):253-61. doi: 10.1016/j.jmb.2012.01.042. Epub 2012 Jan 30.
Flexible and fully disordered protein regions that fold upon binding mediate numerous protein-protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a β-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the β-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the β-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the β-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.
能够灵活折叠的无规蛋白区域可介导众多蛋白-蛋白相互作用。然而，对于其相互作用机制却知之甚少。神经元型一氧化氮合酶（nNOS）的一个柔性区域在与蛋白配体（PSD-95 上的 PDZ[PSD-95（突触后密度蛋白-95）/Discs large/ZO-1]结构域）结合时会发生这种折叠和结合耦联现象。我们通过蛋白质工程与动力学实验的结合分析了该结合反应。β-指结构的突变使蛋白的解离速率常数发生了主要变化，对结合速率常数的影响较小。因此，突变影响了折叠和结合耦联反应的后期事件。我们的结果表明，在结合的限速转变态中不存在β-指的天然结合相互作用，而是以协同的方式在下降沿形成。然而，通过突变，我们可以进一步使β-指失稳，并改变限速步骤，使得初始构象变化成为限速步骤。这种限速步骤的转变表明，在蛋白质的柔性和无规区域中可能存在多步结合机制。
