Characterization of ligand-binding domains and the ligand binding site on protein disulphide-isomerase

蛋白质二硫键异构酶上配体结合域和配体结合位点的表征

• 批准号: BB/D017807/1
• 负责人: Robert Freedman
• 金额: $ 38.43万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD017807%2F1
• 关键词: Characterization; ligand; binding; domains; site

Proteins carry out most key biological activities in all organisms, and the individual biological activity of each protein is crucially dependent on its specific 3-dimensional structure. One of the most significant discoveries in cell biology in the past 20 years has been the recognition that there is an extensive and complex machinery in cells devoted to ensuring that newly-made proteins fold up and assemble correctly to their specific and unique 3-dimensional structure. This machinery comprises 'folding catalysts' which ensure that proteins fold rapidly and correctly and 'molecular chaperones' which prevent misfolding. This machinery is now quite well-defined -- in terms of the identification of its component parts -- but we know very little about how these folding catalysts and chaperones work in molecular detail. The aim of this proposal is to increase our detailed molecular-level understanding of one part of the cellular protein folding machinery. Protein disulphide-isomerase (PDI) is a folding catalyst and chaperone which has been known for many years. It is absolutely required for the folding and assembly of proteins that contain disulphide bonds. Disulphide bonds provide proteins with additional stability and are found in almost all proteins which are secreted from cells or exposed at the extracellular surface of cells. Since this group of proteins includes most protein hormones and other intercellular messengers, hormone receptors, digestive enzymes, antibodies, blood clotting proteins, and (in other species) venom toxins, plant storage proteins etc., this is a very significant class of proteins. For example, most of the human protein drugs which are currently used in therapy (such as insulin, interferons, growth hormones, antibody fragments, blood clotting factors etc). are disulphide-bonded proteins. To the best of our knowledge, PDI or a closely related member of the PDI family, is required for the correct folding of all such proteins. Consequently, more detailed understanding of PDI would be significant not only for basic cell biology but also for medical, veterinary and biotechnological applications. Surprisingly, after almost 30 years of study, the detailed structure of PDI is not known at the molecular level, and so we cannot picture precisely how it acts to assist newly-made proteins to fold and form correct disulphide bonds. There appear to be some difficulties which have frustrated conventional approaches using x-ray crystallography. Preliminary work that we and our collaborators have done over the past 2-3 years suggests that we now understand the basis of these difficulties and makes it possible to plan how to determine the structure of PDI bit-by-bit. We plan to start with the 'domain' of PDI which is most interesting to us, because we know that it is the key domain for the 'chaperone' properties of PDI. We will determine the structure of this domain alone and in combination with a neighbouring domain, as a step towards determining the whole structure. We will not focus simply on a static picture but also determine the flexibility of these parts of PDI, in order to picture the range of molecular motions they undergo on various timescales. Finally we will study these domains of PDI in combination with some small proteins and even smaller fragments (peptides) aiming to understand how PDI and the proteins on which it acts bind to each other and how each influences the detailed structure and dynamics of the other. This will finally give us some insight into how PDI works, in molecular terms.

蛋白质在所有生物体中执行大多数关键的生物活性，每种蛋白质的个体生物活性至关重要地依赖于其特定的三维结构。在过去的20年里，细胞生物学中最重要的发现之一是认识到细胞中存在着一种广泛而复杂的机制，致力于确保新生成的蛋白质折叠并正确地组装成它们特定而独特的三维结构。这种机制包括确保蛋白质快速正确折叠的“折叠催化剂”和防止错误折叠的“分子伴侣”。就其组成部分的识别而言，这种机制现在已经非常明确了，但我们对这些折叠催化剂和伴侣蛋白在分子细节上是如何工作的知之甚少。这项提议的目的是增加我们对细胞蛋白质折叠机制的一部分的详细分子水平的理解。蛋白质二硫异构酶（PDI）是一种折叠催化剂和分子伴侣，已被人们认识多年。它对于含有二硫键的蛋白质的折叠和组装是绝对必需的。二硫键为蛋白质提供了额外的稳定性，几乎所有从细胞分泌或暴露在细胞外表面的蛋白质中都存在二硫键。由于这组蛋白质包括大多数蛋白质激素和其他细胞间信使，激素受体，消化酶，抗体，凝血蛋白，以及（在其他物种中）毒液毒素，植物储存蛋白等，这是一类非常重要的蛋白质。例如，目前用于治疗的大多数人类蛋白质药物（如胰岛素、干扰素、生长激素、抗体片段、凝血因子等）。是二硫化物键合的蛋白质。据我们所知，PDI或PDI家族的一个密切相关的成员是所有这些蛋白质正确折叠所必需的。因此，更详细地了解PDI不仅对基本细胞生物学，而且对医学、兽医和生物技术应用都具有重要意义。令人惊讶的是，经过近30年的研究，PDI在分子水平上的详细结构尚不清楚，因此我们无法准确描绘它是如何帮助新合成的蛋白质折叠并形成正确的二硫键的。使用x射线晶体学的传统方法似乎遇到了一些困难。我们和我们的合作者在过去2-3年里所做的初步工作表明，我们现在了解了这些困难的基础，并有可能计划如何逐位确定PDI的结构。我们计划从PDI的“域”开始，这是我们最感兴趣的，因为我们知道它是PDI“伴侣”属性的关键域。我们将单独确定这个域的结构，并结合邻近的域，作为确定整个结构的一步。我们不会简单地关注静态图像，还将确定PDI这些部分的灵活性，以便描绘它们在不同时间尺度上经历的分子运动范围。最后，我们将研究PDI的这些结构域与一些小蛋白质甚至更小的片段（肽）的结合，旨在了解PDI及其作用的蛋白质如何相互结合，以及每种蛋白质如何影响彼此的详细结构和动力学。这将最终让我们从分子的角度了解PDI是如何工作的。

项目成果

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

• DOI: 10.1371/journal.pone.0082511
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Irvine AG;Wallis AK;Sanghera N;Rowe ML;Ruddock LW;Howard MJ;Williamson RA;Blindauer CA;Freedman RB
• 通讯作者: Freedman RB

Plasticity of human protein disulfide isomerase: evidence for mobility around the X-linker region and its functional significance.

• DOI: 10.1074/jbc.m110.107839
• 发表时间: 2010-08-27
• 期刊: The Journal of biological chemistry
• 作者: Wang C;Chen S;Wang X;Wang L;Wallis AK;Freedman RB;Wang CC
• 通讯作者: Wang CC