# Evolution of fold-switching in the metamorphic chemokine XCL1

> **NIH NIH R56** · MEDICAL COLLEGE OF WISCONSIN · 2021 · $390,000

## Abstract

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.

## Key facts

- **NIH application ID:** 10475442
- **Project number:** 1R56AI155881-01A1
- **Recipient organization:** MEDICAL COLLEGE OF WISCONSIN
- **Principal Investigator:** Brian F Volkman
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $390,000
- **Award type:** 1
- **Project period:** 2021-09-14 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475442

## Citation

> US National Institutes of Health, RePORTER application 10475442, Evolution of fold-switching in the metamorphic chemokine XCL1 (1R56AI155881-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10475442. Licensed CC0.

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