Evolution of fold-switching in the metamorphic chemokine XCL1

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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.