Mapping and Exploiting the Internal Wiring of Dynamic Protein Structures

项目摘要/摘要 变构是一种信号从蛋白质结构的一个部分传递到另一个部分的过程，它在 调节许多生物过程。尽管别构传统上与低聚物有关，但现在它 被认为基本上是所有蛋白质的固有属性。然而，我们对此的基本理解 变构仍然不完整。这一差距使我们无法测试变构的机械假说。 生物医学重要蛋白如动态蛋白酪氨酸磷酸酶的功能调节 酵素。100多个人类PTP在信号转导和疾病中发挥着关键的、多样的和特定的作用， 与糖尿病、癌症或神经疾病有关的各种个体PTP。然而，与 补充蛋白酪氨酸激酶，目前还没有被批准的药物来治疗相对较少的PTPs。 我最近的工作在理解PTP规则方面开辟了新的天地，揭示了令人惊讶的广泛 原型PTP家族成员PTP1B催化结构域中的变构网络 小分子的扰动或从连续的调节界面到动态活性部位的突变 以抑制催化作用。在一个PTP研究结果的基础上，我的团队将探索变构网络是如何 连接在不同PTP的动物园中，它们都有非常相似的催化结构域，与高度相互作用 不同的监管领域。我们将研究一组精选的8-10名PTP，他们被选为与治疗相关的患者， 实验易操纵性和序列多样性，包括但不限于TCPTP、CD45、STEP、LYP和 Shp2.为了映射这些蛋白质中变构背后的相关构象变化，我们将开发 X射线结晶学中使用微扰序列(温度，压力， 湿度等)为了在蛋白质中诱导和模拟机械反应，这种机械反应是远程连接的基础 功能站点。为了阐明这些特定的变构特征是如何在进化过程中产生的，尽管 限制所有PTP共享的催化结构域，我们将创建催化和 来自不同PTP的监管领域，并探索每个领域必须为另一个定制的程度 利用蛋白质设计计算和筛选实验。这些结果将为我们提供关于如何 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，而且在 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 关于个别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产生新的 为特定的治疗发展提供立足点。总的来说，拟议的工作将对以下方面产生广泛影响 生物物理学中的基本问题，包括构象系综如何驱动蛋白质的功能，以及 打开变构治疗开发和蛋白质工程的新大门。

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.