Evolution of fold-switching in the metamorphic chemokine XCL1

项目总结/摘要 这个项目的目标是了解如何以及为什么变性蛋白质从非变性蛋白质进化而来。 使用人类趋化因子XCL 1作为模型系统的变形祖先。几乎所有已知的蛋白质 采用单一折叠结构，但XCL 1是一个罕见的折叠转换或变形蛋白质的例子。 变质蛋白质在两种完全不同的、不相容的结构之间可逆地交换。 因为折叠转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，XCL 1可能从非变性的 （“单态”）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原型变性蛋白XCL 1。目标1中的实验是 旨在检验XCL 1蛋白质祖先中的二硫键丢失伴随着 保留趋化因子折叠同时允许折叠转换突变的其他允许突变 这是为了积累。利用祖先序列重建和核磁共振光谱，我们将复活 比较XCL 1中分支点序列的结构和折叠转换行为 进化我们希望确定关键的突变，赋予变形折叠， 单态XCL 1祖先。具体目标2试图回答这个问题：为什么人类XCL 1 变态的？我们假设折叠转换赋予XCL 1功能优势， 它的祖先，随后优化其在人类免疫系统中的作用。XCL 1结合， 使用保守的趋化因子折叠激活趋化因子受体XCR 1。然而，我们最近 确定了另一种受体，结合其替代非趋化因子折叠，这种相互作用可能具有 对XCL 1进化施加了选择性压力，并将通过以下方式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 在两个不同的折叠的单体结构之间转换。最有前途的结构动力学 设计将通过NMR和其他生物物理测量来表征。变形设计和 相关的单形层序将进行系统分析，以评估 接口优化、灵活性或应变以及内部接触网络，并确定 在一个蛋白质中编码多种结构。总体而言，拟议的研究将提供更深入的 了解折叠开关蛋白的进化起源，这是一个重要但代表性不足的问题。 一类生物分子。

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.