Evolution and design of metamorphic fold-switching proteins

变态折叠转换蛋白的进化和设计

• 批准号: 10733814
• 负责人: Brian F Volkman
• 金额: $ 62.64万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-16 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733814
• 关键词: Adopted; Affinity; Animals; Binding; Biological; Biological Models; Biomedical Technology; Breathing; Categories; Cell surface; Chemicals; Chemotaxis; Computing Methodologies; Dendritic Cells; Engineering; Equilibrium; Evolution; Extracellular Matrix; Family; G-Protein-Coupled Receptors; Glycosaminoglycans; Goals; Health; Human; Immune system; Knowledge; Leukocyte Trafficking; Locomotion; Machine Learning; Maps; Measures; Methods; Molecular Conformation; Mutation; NMR Spectroscopy; Nanotechnology; Nature; Physics; Physiological; Proteins; Recording of previous events; Role; Structure; Structure-Activity Relationship; Testing; Thermodynamics; XCL1 gene; XCR1 gene; antimicrobial; beta pleated sheet; biophysical properties; chemokine; chemokine receptor; design; dimer; disulfide bond; experimental study; flexibility; improved; monomer; novel; optical sensor; pressure; protein folding; receptor; receptor binding; reconstruction; thermostability; tool

Project Summary/Abstract The goals of this project are to understand how and why a metamorphic protein evolved from a non- metamorphic ancestor and develop a method for de novo design of fold-switching proteins. 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. Experiments proposed in specific aim 1 seek 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 discovered that an atypical receptor binds the non-chemokine fold, and may have exerted selective pressure on XCL1 evolution. We will define the structural basis for XCL1 recognition by both receptors. We recently used ancestral sequence reconstruction to show that XCL1 evolved from a non- metamorphic chemokine fold and identified the sequence changes that permitted spontaneous interconversion with an unrelated beta sheet structure. Specific aim 2 will define structure-function relationships for the resurrected XCL1 ancestors and test the hypothesis that cold denaturation of the beta sheet structure enables interconversion with the chemokine fold. In specific aim 3, we will use Rosetta and AlphaFold2 to identify novel sequences that shift between two distinct, folded, monomeric helical bundle 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 and design of fold-switching proteins, an important but underrepresented category of biomolecules.

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。 变形的祖先，并开发一种方法从头设计的折叠开关蛋白质。几乎所有 已知的蛋白质采用单一折叠结构，但XCL 1是折叠转换的罕见例子，或 变质蛋白质变态蛋白质在两种完全不同的， 不兼容的结构。在具体目标1中提出的实验试图回答这个问题： 人类XCL1变形？我们假设，折叠转换赋予了一个功能优势， XCL 1祖先随后针对其在人类免疫系统中的作用进行了优化。XCL1结合 并使用保守的趋化因子折叠激活趋化因子受体XCR 1。但我们 发现一种非典型受体结合非趋化因子折叠， XCL1进化的压力。我们将定义两种受体识别XCL 1的结构基础。 我们最近使用祖先序列重建来证明XCL1是从一个非- 变形趋化因子折叠，并确定了允许自发的序列变化， 与不相关的β折叠结构相互转化。具体目标2将定义结构-功能 复活的XCL 1祖先的关系，并测试假设，冷变性的 β折叠结构能够与趋化因子折叠相互转化。在具体目标3中，我们将使用 Rosetta和AlphaFold2鉴定在两个不同的折叠单体之间转移的新序列 螺旋束结构最有前途的设计的结构动力学特征将是 NMR和其他生物物理测量。变质设计和有关的单形序列 将进行系统分析，以评估接口优化、灵活性或 应变和内部接触网络，以及识别在一个 单一蛋白质总的来说，拟议的研究将使人们更深入地了解 折叠开关蛋白的进化起源和设计，一个重要但代表性不足的类别， 生物分子