Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured ¹⁵N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 μs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

译文

:固有的无序蛋白(IDP)通常依靠静电相互作用来结合其结构化靶标。为了深入了解静电相遇复合物的形成机理,我们研究了肽Sos(PPPVPPRRRR)作为IDP的最小模型,与c-Crk N末端SH3结构域的结合。最初,我们在两个磁场强度下测量了1 N的弛豫率,并确定了Sos与野生型SH3的复合物的结合位移。我们还使用琥珀色ff99SB * -ILDN力场记录了该复合物的3μs分子动力学(MD)轨迹。实验数据和模拟数据的比较表明,MD模拟始终高估了结合界面上盐桥相互作用的强度。使用其他先进的力场进行的一系列模拟也未能产生任何令人满意的结果。为解决此问题,我们对Affff ff99SB * -ILDN琥珀色力场进行了经验校正,据此得出了横跨Arg-to-Asp和Arg-to-Glu盐桥的氮氧对的Lennard-Jones平衡距离增加了3%。实施此校正可在仿真和实验之间取得良好的一致性。调节盐桥相互作用的强度去除了原始MD模型中包含的一定量的应变,从而改善了肽的疏水性N-末端部分的结合。发现该肽的富含精氨酸的C-末端部分不受过度稳定的盐桥的影响,可以在其多种构象状态之间更快速地相互转化。修改后的MD协议也已成功用于模拟整个绑定过程。为此,首先将肽放置在蛋白质表面上方的较高位置。然后,它到达晶体学坐标约2Å以内的正确束缚姿势。这种模拟使我们能够分析动态结合中间体的细节,即静电相遇复合物。但是,这种瞬态,人口稀少状态的实验表征仍然遥不可及。为了克服这个问题,我们设计了c-Crk N-SH3的双突变体,其中突变Y186L和W169F消除了紧密的Sos结合,并使平衡向类似于静电相遇复合物的中间态移动。此工程系统的NMR和MD结合研究的结果将在本文的下一部分中报告。

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