Evolution and design of metamorphic fold-switching proteins

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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.