Using dynamic network models to quantitatively predict changes in binding affinity/specificity that arise from long-range amino acid substitutions

总结 先进的测序技术提供了越来越多的关于人类遗传变异的数据 和病毒进化。然而，预测蛋白质编码区错义突变的结果仍然是 这是一个挑战，在区分生物医学相关变异与中性变异（几乎没有或 对表型没有影响）。特别是，当错义突变改变了氨基酸序列时， 远离蛋白质功能/结合位点的酸。这些缺点也损害蛋白质 设计我们建议通过开发定量的计算模型来改善这些需求， 长距离取代对结合相互作用的影响。为此，我们制定了一项办法， 其中（1）蛋白质的集体运动首先通过分子动力学模拟揭示，然后（2）力 扰动用于破坏蛋白质的平衡，从而近似配体结合的效果。我们 我在已发表的研究和初步数据中使用了这种方法来阐明动力学的传播 通过蛋白质的各向异性相互作用网络发生变化。结果表明，这些动态变化 网络对蛋白质功能有至关重要的影响，从而导致我们的中心假设：长- 配体结合上的距离取代是蛋白质动态偶联的变化的紧急性质， 各向异性网络当前提案的目标是扩展这种计算方法， 模型预测：（目标1）由长距离，调节 （目的2）哪些非接触取代对结合亲和力具有非加和效应 （“上位性”）;和（目的3）哪些长距离位置有助于配体特异性。为此，我们有一个 良好的合作，使我们能够在计算预测和实验之间进行权衡。 测试，能够开发具有计算精度的定量模型。我们的初步研究 E.大肠杆菌乳糖阻遏蛋白（LacI），实验结果验证了我们的 初步计算模型，并为目标1-3提供具体假设。另外的模型蛋白将 用于显示我们的方法的一般性，并将包括LacI同系物PurR，cAMP受体 蛋白和病毒蛋白酶SARS-Cov 2-Mpro。结果将用于提供新的计算工具， 预测远距离替代的功能结果。该项目的成功将促进研究 在蛋白质结构生物学，分子遗传学，进化和医学的接口，通过推进 对远离功能位点的取代如何改变配体结合的机制的理解。

Summary Advanced sequencing technologies provide ever-increasing quantities of data about human genetic variation and viral evolution. However, predicting the outcomes of missense mutations in protein coding regions remains a challenge, creating a bottleneck in discriminating biomedically-relevant variants from neutral ones (with little or no effect on phenotype). In particular, outcome predictions are very poor when a missense mutation alters amino acids that are located far from a protein’s functional/binding sites. These shortcomings also impair protein design. We propose to ameliorate these needs by developing quantitative, computational models that predict the effects of long-distance substitutions on binding interactions. To that end, we have developed an approach in which (1) a protein’s collective motions are first revealed by molecular dynamics simulations and then (2) force perturbation is used to disrupt the protein’s equilibrium, thereby approximating the effects of ligand binding. We have used this approach in published studies and preliminary data to illuminate the propagation of dynamical changes through a protein’s anisotropic network of interactions. Results suggest that changes in these dynamic networks have crucial effects on protein function, thereby leading to our central hypothesis: The effects of long- distance substitutions on ligand binding are emergent properties of changes in the protein’s dynamically-coupled, anisotropic network. The goal of the current proposal is to extend this computational approach to develop models that predict: (Aim 1) the magnitudes of binding affinity changes arising from long-distance, modulating substitutions; (Aim 2) which pairs of non-contact substitutions have non-additive effects on binding affinities (“epistasis”); and (Aim 3) which long-distance positions contribute to ligand specificity. To that end, we have a well-established collaboration that allows us to iterate between computational predictions and experimental testing, enabling development of quantitative models with computed accuracies. Our preliminary studies used the well-characterized E. coli lactose repressor protein (LacI), for which experimental results validate our preliminary computational models and provide specific hypotheses for Aims 1-3. Additional model proteins will be used to show the generality of our approach and will include the LacI homolog PurR, the cAMP receptor protein, and a viral protease SARS-Cov2-Mpro. Results will be used to provide novel computational tools for predicting functional outcomes of long-distance substitutions. The success of this project will catalyze research at the interface of protein structural biology, molecular genetics, evolution and medicine by advancing the mechanistic understanding of how substitutions distal from functional sites alter ligand binding.