Evolution of fold-switching in the metamorphic chemokine XCL1

项目摘要/摘要 这个项目的目标是了解一种变质蛋白质是如何以及为什么从非 使用人类趋化因子XCL1作为模型系统的变质祖先。几乎所有已知的蛋白质 采用单一折叠结构，但XCL1是折叠切换或变性蛋白质的罕见例子。 变质蛋白质在两个完全不同的、不相容的结构之间可逆地交换。 因为折叠切换与两个绝对保守的二硫键不相容 在趋化因子家族的其他地方，XCL1很可能是从一个非变质的 (‘单态的’)祖先。我们建议研究褶皱的演化和化学控制。 在三个特定的目标中切换到原型变质蛋白XCL1。目标1中的实验是 旨在测试XCL1的蛋白质祖先中二硫键缺失伴随着 其他允许的突变，在保留趋化因子折叠的同时允许折叠切换突变 那就是积累。利用祖先序列重建和核磁共振波谱，我们将复活 并比较了XCL1中分支点序列的结构和折叠切换行为 进化论。我们希望找出导致变性折叠的关键突变。 单态XCL1祖先。《特定目的2》试图回答这个问题：为什么人类XCL1 变态？我们假设折叠切换为XCL1提供了功能优势 它的祖先后来被优化了它在人类免疫系统中的作用。XCL1绑定和 利用保守的趋化因子折叠激活趋化因子受体XCR1。然而，我们最近 发现了另一个与其替代的非趋化因子折叠结合的受体，这种相互作用可能具有 对XCL1的进化施加了选择性压力，并将定义其识别的结构基础 两种受体蛋白。在具体目标3中，我们将使用Rosetta多态设计来识别序列 两个截然不同的折叠单体结构之间的转换。最有前途的结构动力学 设计将通过核磁共振和其他生物物理测量来表征。变形的设计和 相关的单态序列将被系统地分析，以评估 接口优化、灵活性或紧张性以及内部联系网络，并确定需要的功能 在一种蛋白质中编码多种结构。总的来说，拟议的研究将提供更深层次的 了解折叠转换蛋白的进化起源，这是一种重要但未被充分代表的蛋白质 生物分子的范畴。

Project Summary/Abstract The goal of this project is to understand how and why a metamorphic protein evolved from a non- metamorphic ancestor using the human chemokine XCL1 as a model system. Nearly all known proteins adopt a single folded structure, but XCL1 is a rare example of a fold-switching, or metamorphic, protein. Metamorphic proteins reversibly exchange between two entirely different, incompatible structures. Because fold-switching is incompatible with the two disulfide bonds that are absolutely conserved elsewhere in the chemokine family, XCL1 likely evolved to be metamorphic from a non-metamorphic (`monomorphic') ancestor. We propose to investigate the evolution and chemical control of fold- switching in the prototypical metamorphic protein XCL1 in three specific aims. Experiments in aim 1 are designed to test the hypothesis that disulfide loss in a protein ancestor of XCL1 was accompanied by other permissive mutations that preserved the chemokine fold while allowing fold-switching mutations that to accumulate. Using ancestral sequence reconstruction and NMR spectroscopy, we will resurrect and compare the structures and fold-switching behavior of the sequences at branch points in XCL1 evolution. We expect to identify key mutations that imparted metamorphic folding to the monomorphic XCL1 ancestor. Specific aim 2 seeks to answer the question: why is human XCL1 metamorphic? We hypothesize that fold-switching conferred a functional advantage to an XCL1 ancestor that was subsequently optimized for its role in the human immune system. XCL1 binds and activates the chemokine receptor XCR1 using the conserved chemokine fold. However, we recently identified another receptor that binds its alternative non-chemokine fold, an interaction that may have exerted selective pressure on XCL1 evolution, and will define the structural basis for its recognition by both receptor proteins. In specific aim 3, we will use Rosetta multi-state design to identify sequences that shift between two distinct, folded, monomeric structures. Structural dynamics of the most promising designs will be characterized by NMR and other biophysical measurements. Metamorphic designs and related monomorphic sequences will be systematically analyzed to assess the relative importance of interface optimization, flexibility or strain, and internal contact networks and identify features required to encode multiple structures in a single protein. Collectively, the proposed studies will provide a deeper understanding of the evolutionary origin of fold-switching proteins, an important but underrepresented category of biomolecules.