Molecular recognition in the streptavidin-biotin system

描述(申请人提供)：链霉亲和素/生物素复合体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。对链霉亲和素/生物素复合体的高分辨晶体结构的分析揭示了许多紧密结合的复合体的良好的蛋白质-配体接触。然而，我们对大量链霉亲和素突变体的广泛结构和热力学表征强烈表明，直接的蛋白质-配体接触不能完全解释生物素非常紧密的结合。我们以前的研究也表明，生物素在配体结合和解离反应中遵循明确的反应坐标，某些突变可以改变结合/解离反应的活化能垒，而对平衡结构没有任何显著影响。这些结果导致我们提出链霉亲和素的平衡动力学，即结构涨落，有助于确定平衡结合能和激活能垒。我们将通过对野生型和所选突变链霉亲和素/生物素复合体的各向异性温度因子进行分子动力学模拟和详细的结晶学分析来检验这一假设，然后在野生型和突变型复合体中寻找结构波动和量热测量之间的相关性。我们以前的结果也表明，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来测试这一建议，通过进一步表征链霉亲和素结合部位的水相互作用。我们将使用这些模拟结果来建议突变，这些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。这些研究将加深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应该有用的重要概念。这些信息将有助于合理设计治疗上重要的蛋白质靶标的药物。

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.