Molecular recognition in the streptavidin-biotin system

描述（由申请人提供）：链亲和素/生物素复合物涉及生物学中观察到的最强的非共价相互作用之一，是研究高亲和力蛋白质/配体相互作用的理想模型系统。链亲和素/生物素复合物的高分辨率晶体结构分析揭示了紧密结合复合物的许多有利的蛋白质-配体接触特征。然而，我们对大量链亲和素突变体的广泛结构和热力学表征强烈表明，蛋白质与配体的直接接触不能完全解释生物素的紧密结合。我们之前的研究也表明，在配体结合和解离反应中，生物素遵循一个明确的反应坐标，某些突变可以改变结合/解离反应的活化能势垒，而对平衡结构没有明显影响。这些结果使我们提出链霉亲和素平衡动力学或结构波动有助于确定平衡结合能和活化能势垒。我们将通过分子动力学模拟和对野生型和选择突变型链霉亲和素/生物素复合物的各向异性温度因子进行详细的晶体学分析来验证这一假设，然后寻找野生型和突变型复合物的结构波动和量热测量之间的相关性。我们之前的研究结果也表明，特定的水分子在生物素结合/解离反应中起着关键的能量作用。我们将使用分子动力学模拟，通过进一步表征链霉亲和素结合位点的水相互作用来验证这一提议。我们将使用这些模拟结果来提示突变可以改变络合物中的平衡水行为，从而调节配体结合自由能和/或活化能。这些研究将增强我们对高亲和力蛋白质/配体结合相互作用的理解，并将帮助我们阐明在基于结构的配体设计项目中有用的重要概念。这些信息将有助于合理的药物设计应用于治疗重要的蛋白质靶点。

DESCRIPTION (provided by applicant): The streptavidin/biotin complex involves one of the strongest non-covalent interactions observed in biology, and is an ideal model system for the study of high-affinity protein/ligand interactions. Analysis of high- resolution crystal structures for the streptavidin/biotin complex reveals numerous favorable protein-ligand contacts characteristic of a tightly bound complex. However, our extensive structural and thermodynamic characterization of numerous streptavidin mutants strongly suggests that direct protein-ligand contacts cannot fully explain the extremely tight biotin binding. Our previous studies also suggest that biotin follows a well defined reaction coordinate during ligand binding and dissociation reactions, and that certain mutations can alter the activation energy barrier for the binding/dissociation reactions, without any significant effect on the equilibrium structure. These results lead us to propose that streptavidin equilibrium dynamics, or structural fluctuations, help determine the equilibrium binding energy and activation energy barrier. We will test this hypothesis by performing molecular dynamics simulations and detailed crystallographic analyses of anisotropic temperature factors for wild-type and selected mutant streptavidin/biotin complexes, and then look for correlations between structural fluctuations and calorimetric measurements in wild-type versus mutant complexes. Our previous results also suggest that specific water molecules play a key energetic role in the biotin binding/dissociation reactions. We will use molecular dynamics simulations to test this proposal by further characterizing water interactions in the streptavidin binding site. We will use these simulation results to suggest mutations that can alter the equilibrium water behavior in the complex, and thus modulate the ligand binding free energy and/or activation energy. These studies will enhance our understanding of high-affinity protein/ligand binding interactions, and will help us elucidate important concepts that should be useful in structure-based ligand design projects. Such information will be helpful in rational drug design applications for therapeutically important protein targets.