# Evolution and design of metamorphic fold-switching proteins

> **NIH NIH R01** · MEDICAL COLLEGE OF WISCONSIN · 2024 · $554,302

## Abstract

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.

## Key facts

- **NIH application ID:** 10870002
- **Project number:** 5R01AI168423-02
- **Recipient organization:** MEDICAL COLLEGE OF WISCONSIN
- **Principal Investigator:** Brian F Volkman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $554,302
- **Award type:** 5
- **Project period:** 2023-06-16 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10870002

## Citation

> US National Institutes of Health, RePORTER application 10870002, Evolution and design of metamorphic fold-switching proteins (5R01AI168423-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10870002. Licensed CC0.

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