Mapping and Exploiting the Internal Wiring of Dynamic Protein Structures

项目概要/摘要 信号从蛋白质结构的一个部分传递到另一个部分，在蛋白质结构中起着关键作用。 调节许多生物过程。虽然变构在传统上与低聚物有关，但现在 被认为是基本上所有蛋白质的固有属性。然而，我们对 变构仍然不完全。这一差距使我们无法验证关于变构的机制假说。 调节生物医学重要蛋白质的功能，如动态蛋白酪氨酸磷酸酶 内切酶100多个人类PTP在信号转导和疾病中发挥着关键、多样和特异的作用， 与糖尿病、癌症或神经系统疾病相关的各种PTPs。但不同于 由于蛋白酪氨酸激酶是一种互补的蛋白酪氨酸激酶，因此没有批准用于相对研究不足的PTP的药物。 我最近的工作在理解PTP调节方面开辟了新的天地，揭示了一个令人惊讶的广泛的 在原型PTP家族成员PTP 1B的催化结构域中的变构网络， 小分子干扰或从连续调节界面到动态活性位点的突变 以抑制催化作用。基于一个PTP的这一结果，我的团队将探索变构网络是如何“重新”的。 在不同PTP的动物园中，它们都具有非常相似的催化结构域， 不同的监管领域。我们将研究一组8-10名经选择的治疗相关性PTP， 实验易处理性和序列多样性，包括但不限于TCPTP、CD 45、STEP、LYP和 SHP2.为了绘制这些蛋白质变构的相关构象变化，我们将开发 X射线晶体学中的新的多数据集方法使用扰动系列（温度，压力， 湿度等）在蛋白质中引发和模拟机械反应，该反应是远程之间耦合的基础， 功能性网站为了阐明这些特定的变构特征是如何在进化过程中出现的， 限制的催化结构域架构共享的所有PTP，我们将创建嵌合体的催化和 监管领域从不同的PTP，并探讨在何种程度上必须定制的其他 使用蛋白质设计计算和选择实验。研究结果将为我们提供关于 模块化蛋白质可以重组以实现正交功能，不仅在自然进化中， 在合成生物学中创造生物传感器或计算分子电路。这项工作将导致假设 关于个体PTP中独特的变构弱点，我们将使用新的高通量X射线- 基于小分子筛选方法和后续化学生物学实验， 为特定的治疗发展奠定基础。总的来说，拟议的工作将对以下方面产生广泛影响： 生物物理学的基本问题，包括构象集合如何驱动蛋白质功能，并将 为变构治疗开发和蛋白质工程打开了新的大门。

Project Summary/Abstract Allostery, in which a signal is transmitted from one part of a protein structure to another, plays critical roles in regulating many biological processes. Although allostery is traditionally associated with oligomers, it is now thought to be an inherent property of essentially all proteins. However, our fundamental understanding of allostery remains incomplete. This gap prevents us from testing mechanistic hypotheses about allosteric regulation of function in biomedically important proteins such as the dynamic Protein Tyrosine Phosphatase enzymes. The 100+ human PTPs play critical, diverse, and specific roles in signal transduction and diseases, with various individual PTPs linked to diabetes, cancers, or neurological disorders. However, unlike the complementary Protein Tyrosine Kinases, there are no approved drugs for the relatively understudied PTPs. My recent work broke new ground in understanding PTP regulation by revealing a surprisingly extensive allosteric network in the catalytic domain of the archetypal PTP family member, PTP1B, that transmits perturbations by small molecules or mutations from a contiguous regulatory interface to the dynamic active site to inhibit catalysis. Building off this result for one PTP, my group will explore how allosteric networks are “re- wired” in a menagerie of different PTPs, which all have very similar catalytic domains that interact with highly varied regulatory domains. We will study a select group of 8-10 PTPs selected for therapeutic relevance, experimental tractability, and sequence diversity, including but not limited to TCPTP, CD45, STEP, LYP, and SHP2. To map the correlated conformational changes that underlie allostery in these proteins, we will develop new multidataset approaches in X-ray crystallography that use perturbation series (temperature, pressure, humidity, etc.) to elicit and model mechanical responses in the protein that underlie coupling between remote functional sites. To elucidate how these specific allosteric signatures arose during evolution despite the constraints of a catalytic domain architecture shared by all PTPs, we will create chimeras of catalytic and regulatory domains from different PTPs, and explore the extent to which each must be customized for the other using protein design calculations and selection experiments. The results will provide general insights into how modular proteins can be recombined to achieve orthogonal functions, not only in natural evolution but also for creating biosensors or computational molecular circuits in synthetic biology. This work will lead to hypotheses about unique allosteric weak points in individual PTPs, which we will test using new high-throughput, X-ray- based small-molecule screening methods and followup chemical biology experiments to generate new footholds for specific therapeutic development. Overall, the proposed work will have broad implications for fundamental questions in biophysics, including how conformational ensembles drive protein function, and will open new doors in allosteric therapeutic development and protein engineering.