Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.

Autor:	Caldwell SJ; Department of Biochemistry, University of Washington, Seattle, WA 98195.; Institute for Protein Design, University of Washington, Seattle, WA 98195., Haydon IC; Department of Biochemistry, University of Washington, Seattle, WA 98195.; Institute for Protein Design, University of Washington, Seattle, WA 98195., Piperidou N; Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland., Huang PS; Department of Biochemistry, University of Washington, Seattle, WA 98195.; Institute for Protein Design, University of Washington, Seattle, WA 98195.; Department of Bioengineering, Stanford University, Shriram Center for Bioengineering and Chemical Engineering, Stanford, CA 94305., Bick MJ; Department of Biochemistry, University of Washington, Seattle, WA 98195.; Institute for Protein Design, University of Washington, Seattle, WA 98195., Sjöström HS; Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland., Hilvert D; Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland., Baker D; Department of Biochemistry, University of Washington, Seattle, WA 98195; dabaker@uw.edu cathleen.zeymer@tum.de.; Institute for Protein Design, University of Washington, Seattle, WA 98195.; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195., Zeymer C; Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland; dabaker@uw.edu cathleen.zeymer@tum.de.; Department of Chemistry, Technische Universität München, 85747 Garching, Germany.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Dec 01; Vol. 117 (48), pp. 30362-30369. Date of Electronic Publication: 2020 Nov 17.
DOI:	10.1073/pnas.2008535117
Abstrakt:	De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry-a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel-such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts. Competing Interests: The authors declare no competing interest. (Copyright © 2020 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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