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.

项目概要/摘要 该项目的目标是了解变态蛋白如何以及为何从非变态蛋白进化而来。 变态祖先并开发了一种从头设计折叠转换蛋白的方法。几乎所有 已知的蛋白质采用单折叠结构，但 XCL1 是折叠转换的罕见例子，或者 变质, 蛋白质.变质蛋白在两种完全不同的、 不相容的结构。具体目标 1 中提出的实验试图回答这个问题：为什么 人类XCL1变质？我们假设折叠转换赋予了功能优势 XCL1 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 XCR1。然而，我们 发现非典型受体结合非趋化因子折叠，并可能发挥选择性 XCL1进化的压力。我们将定义两种受体识别 XCL1 的结构基础。 我们最近使用祖先序列重建来表明 XCL1 是从非 变质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折叠结构的相互转化。具体目标 2 将定义结构-功能 复活的 XCL1 祖先的关系并检验冷变性的假设 β片层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 识别在两个不同的折叠单体之间转换的新序列 螺旋束结构。最有前途的设计的结构动力学特点是 核磁共振和其他生物物理测量。变质设计和相关的单态序列 将进行系统分析，以评估界面优化、灵活性或 应变和内部接触网络，并识别编码多个结构所需的特征 单一蛋白质。总的来说，拟议的研究将提供对 折叠转换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。