Developing ex vivo structural biology using natural abundance NMR: the role of conformational dynamics in regulating protein metastability

蛋白质有效地折叠成正确的三维结构对细胞功能至关重要。在大多数情况下，这对应于能量最有利的状态，但一些亚稳态蛋白质折叠成高能构象，这些构象准备在以后需要时根据蛋白质的特定功能进行大规模的结构转换。Serpins就是这样一类亚稳态蛋白，血浆糖蛋白α1抗胰蛋白酶(AAT)就是其中的典型代表。丝氨酸酶是最丰富的蛋白酶抑制剂家族，具有内在的动态分子结构：在抑制底物蛋白酶的过程中，它们经历了从最初的亚稳态构象到形状的戏剧性变化。显然，亚稳性是蛇针功能的核心，构象变化必须能够在需要时有效地触发，但这也是它们的致命弱点：自发转换可能导致错误折叠或形成聚合物聚集体，这一过程通常与疾病有关。在人类发现的35个蛇针基因中，近三分之一已知与遗传病有关，5个已知形成称为聚合物的蛋白质聚集体。然而，尽管进行了多年的研究，但调控这些转化的分子机制仍然知之甚少。我们的假设是，亚稳态结构中的小范围波动(动力学)可能是这个谜题的关键，因此这个项目首先被设计来表征AAT分子的溶液状态结构和动力学，然后将这些观察到的结果与测量的构象变化速率相关联。核磁共振(NMR)光谱是研究蛋白质结构和动力学的一种非常强大的实验技术。然而，核磁共振传统上要求使用专门的同位素标记技术在细菌细胞内重组表达蛋白质，而对于一些感兴趣的分子，包括AAT的几个变体，这目前是不可能的。相反，我们的初步数据颠覆了这一范式，表明我们可以使用直接从人类捐赠者-包括具有罕见的疾病相关突变的患者-提纯的AAT来测量高质量的核磁共振谱，而不需要同位素标记。因此，我们第一次可以研究体外天然糖基化的AAT分子的溶液态结构和动力学，这揭示了一种与疾病相关的变体的构象的广泛变化，这是用将分子限制在刚性晶格结构中的结晶学方法所没有观察到的。我们建议进一步进行这些观察，开发一种新的核磁共振实验工具包，以表征这些未标记的体外蛋白质样本的结构和动力学。我们将详细调查与疾病相关的突变或人工设计的突变对亚稳态蛇针折叠的结构和动力学的影响，并将其与突变对抑制活性以及错误折叠和聚合过程的影响进行比较。通过将AAT变体的溶液结构和动力学与蛇针功能和功能障碍相关联，我们的研究将解决长期存在的问题，即如何调节亚稳态蛋白质的结构变化，这可能最终导致设计错折叠丝针的抑制剂的新的机制基础。更广泛地说，我们将在这个项目中开发的新的核磁共振方法将提供一个平台，可以很容易地扩展到其他以前无法获得的蛋白质系统的体外结构生物学。

The efficient folding of proteins into their correct three-dimensional structures is essential for cellular function. In most cases this corresponds to the energetically most favourable state, but a number of metastable proteins fold instead to high energy conformations, which are primed to undergo large scale structural transformations when later required according to the particular function of the protein. Serpins are one such class of metastable proteins, of which the plasma glycoprotein alpha1-antitrypsin (AAT) is the prototypical example. Serpins comprise the most abundant family of protease inhibitors, and possess a molecular structure that is inherently dynamic: in the process of inhibiting their substrate protease, they undergo a dramatic change in shape from their initial metastable conformation. Clearly then, metastability is central to serpin function and conformational changes must be able to be triggered efficiently when required, and yet it is also their Achilles heel: spontaneous transitions can lead to misfolding or formation of polymeric aggregates, a process that is often associated with disease. Of the 35 serpin genes found in humans, nearly a third have a known involvement in hereditary disease, and five are known to form protein aggregates called polymers. However, despite many years of research the molecular mechanisms by which these transformations can be regulated remain poorly understood. It is our hypothesis that small-scale fluctuations ('dynamics') in the structure of the metastable native state may hold the key to this puzzle, and so this project is designed first to characterise the solution-state structure and dynamics of AAT molecules, and then to correlate these observations with the measured rates of conformational change.Nuclear magnetic resonance (NMR) spectroscopy is an exceptionally powerful experimental technique for studying the structure and dynamics of proteins. However, NMR traditionally requires proteins to be expressed recombinantly within bacterial cells using specialised isotopic labelling techniques, and for a number of interesting molecules, including several variants of AAT, this is not currently possible. Instead, our preliminary data overturn this paradigm by showing we can measure high quality NMR spectra using AAT purified directly from human donors - including patients with rare, disease-associated mutations - without the need for isotopic labelling. Thus, for the first time we can study the solution-state structure and dynamics of ex vivo, natively glycosylated AAT molecules, and this has revealed widespread changes in the conformation of a disease-associated variant that were not observed using crystallographic approaches that confine molecules into a rigid lattice structure. We propose to pursue these observations further, developing a new toolkit of NMR experiments to characterise structure and dynamics in these unlabelled ex vivo protein samples. We will investigate in detail the impact that mutations - associated with disease, or artificially designed - can have upon the structure and dynamics of the metastable serpin fold, and compare this with the effect the mutations have on both inhibitory activity and the misfolding and polymerisation processes.In correlating the solution structure and dynamics of AAT variants with serpin function and dysfunction, our research will address the longstanding problem of how structural changes within metastable proteins can be regulated, and this may ultimately lead to a new mechanistic basis for the design of inhibitors of serpin misfolding. More broadly, the new NMR methodologies that we will develop in this project will provide a platform that can be readily extended to ex vivo structural biology of other previously inaccessible protein systems.