A De Novo Designed Protein with Versatile Metal Binding and Tunable Hydrolytic Activity
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Abstract
Metalloenzyme superfamilies achieve diverse functions within a shared structural framework, and similar functional variety may be achievable in designed proteins. We have previously reported a computational approach that enables the de novo design of symmetric protein assemblies around metal centers with pre-defined coordination geometries. Here, we demonstrate that an artificial protein trimer termed Tet4, whose structure was designed around an idealized tetrahedral His3/H2O-ZnII coordination motif, enables the high-affinity binding of several other divalent first-row transition metal ions in the same geometry as for ZnII. We then follow the proposed evolutionary path of a natural metalloenzyme family by engineering a pseudosymmetric, single chain variant of Tet4, scTet425. scTet425 allows us to introduce asymmetric point mutations that influence the catalytic properties of the metal center. We also demonstrate that we can further tune the enzymatic activity of Tet4 by designing a substrate pocket that improves Zn-Tet4’s affinity for a hydrolysis substrate, 4-methylumbelliferyl acetate.
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- Authors
- Hoffnagle, A. M., Srisantitham, S., Neeley, M., Tsai, C.-Y., & Tezcan, F. A.
- Affiliations
- University of California San Diego (US)
- Corresponding author
- Tezcan, F. Akif
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2025-07-23
- Volume
- 64
- Issue
- 15
- Pages
- 3261-3271
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W4412610193
- Cited by
- 4
- References
- 82
- Citations by year
- 2026: 3 · 2025: 1
- FWCI
- 1.20
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Biochemistry (publishedVersion)
- ChemRxiv (acceptedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (12948080, R01-GM138884)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Keywords
- de novo protein design, metalloenzymes, zinc binding, tetrahedral coordination, protein trimer
- MeSH terms
- Binding Sites, Hydrolysis, Metalloproteins, Metals, Models, Molecular, Protein Binding, Zinc, Protein Engineering
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