Obtaining a detailed description of how active site flap motion affects substrate or ligand binding will advance structure-based drug design (SBDD) efforts on systems including the kinases, HSP90, HIV protease, ureases, etc. Through this understanding, we will be able to design better inhibitors and better proteins that have desired functions. Herein we address this issue by generating the relevant configurational states of a protein flap on the molecular energy landscape using an approach we call MTFlex-b and then following this with a procedure to estimate the free energy associated with the motion of the flap region. To illustrate our overall workflow, we explored the free energy changes in the streptavidin/biotin system upon introducing conformational flexibility in loop3-4 in the biotin unbound ( apo) and bound ( holo) state. The free energy surfaces were created using the Movable Type free energy method, and for further validation, we compared them to potential of mean force (PMF) generated free energy surfaces using MD simulations employing the FF99SBILDN and FF14SB force fields. We also estimated the free energy thermodynamic cycle using an ensemble of closed-like and open-like end states for the ligand unbound and bound states and estimated the binding free energy to be approximately -16.2 kcal/mol (experimental -18.3 kcal/mol). The good agreement between MTFlex-b in combination with the MT method with experiment and MD simulations supports the effectiveness of our strategy in obtaining unique insights into the motions in proteins that can then be used in a range of biological and biomedical applications.

中文翻译：

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活性位点瓣在链霉亲和素/生物素复合物形成中的作用

获得活性位点瓣运动如何影响底物或配体结合的详细描述将推进基于结构的药物设计 (SBDD) 对包括激酶、HSP90、HIV 蛋白酶、脲酶等系统的工作。通过这种理解，我们将能够设计更好的抑制剂和具有所需功能的更好的蛋白质。在这里，我们通过使用我们称为 MTFlex-b 的方法在分子能量图谱上生成蛋白质瓣的相关构型状态来解决这个问题，然后通过一个程序来估计与瓣区域运动相关的自由能。为了说明我们的整体工作流程，我们探索了在生物素未结合 (apo) 和结合 (holo) 状态下在 loop3-4 中引入构象灵活性后链霉亲和素/生物素系统中的自由能变化。自由能表面是使用可移动类型自由能方法创建的，为了进一步验证，我们将它们与使用 FF99SBILDN 和 FF14SB 力场的 MD 模拟产生的自由能表面的平均力 (PMF) 势进行比较。我们还使用配体未结合和结合状态的封闭状和开放状终态集合估计了自由能热力学循环，并估计结合自由能约为 -16.2 kcal/mol（实验性 -18.3 kcal/mol） . MTFlex-b 与 MT 方法与实验和 MD 模拟相结合的良好一致性支持我们的策略在获得对蛋白质运动的独特见解方面的有效性，然后可以将其用于一系列生物和生物医学应用。