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  • Review Article
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De novo metalloprotein design

Nature Reviews Chemistry volume 6, pages 31–50 (2022)Cite this article

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Abstract

Natural metalloproteins perform many functions — ranging from sensing to electron transfer and catalysis — in which the position and property of each ligand and metal are dictated by protein structure. De novo protein design aims to define an amino acid sequence that encodes a specific structure and function, providing a critical test of the hypothetical inner workings of (metallo)proteins. To date, de novo metalloproteins have used simple, symmetric tertiary structures — uncomplicated by the large size and evolutionary marks of natural proteins — to interrogate structure–function hypotheses. In this Review, we discuss de novo design applications, such as proteins that induce complex, increasingly asymmetric ligand geometries to achieve function, as well as the use of more canonical ligand geometries to achieve stability. De novo design has been used to explore how proteins fine-tune redox potentials and catalyse both oxidative and hydrolytic reactions. With an increased understanding of structure–function relationships, functional proteins including O2-dependent oxidases, fast hydrolases and multi-proton/multielectron reductases have been created. In addition, proteins can now be designed using xenobiological metals or cofactors and principles from inorganic chemistry to derive new-to-nature functions. These results and the advances in computational protein design suggest a bright future for the de novo design of diverse, functional metalloproteins.

Key points

  • The metalloprotein designer must first consider the desired function, then select an appropriate active site to achieve it and the tertiary structure to support it.

  • Design constraints can be usefully drawn from either the inorganic chemistry literature or from a bioinformatics approach.

  • There must be a unifying element to the local symmetry of the protein tertiary structure and the metal active site.

  • The design must balance the energetics of protein folding with metal–ligand binding in order to achieve the desired coordination geometry.

  • The introduction of asymmetry is a key strategy for introducing function into metalloproteins and must be compensated for by the introduction of stabilizing elements elsewhere in the design.

  • The design space beyond coiled coils remains sparsely studied and offers opportunities for more diverse active sites and, hence, functions.

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Fig. 1: Three-helix and four-helix bundles.
Fig. 2: Crystal structures of natural metalloproteins illustrating symmetry elements in helical bundles.
Fig. 3: Tripeptide scaffolds for metalloprotein design.
Fig. 4: Asymmetric active sites in three-helix bundles.
Fig. 5: Functional metalloproteins with four-helix bundles.
Fig. 6: Design strategy for well-structured porphyrin-binding proteins.
Fig. 7: β-Sheet-containing designed metalloproteins.

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Acknowledgements

The authors acknowledge support from National Institutes of Health (NIH) grants GMR35 122603, F32GM130029 and F32GM139379. They thank the many coworkers who have contributed to the field, as well as reviewers for helpful comments and suggestions.

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  1. Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA, USA

    Matthew J. Chalkley, Samuel I. Mann & William F. DeGrado

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  1. Matthew J. Chalkley
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  2. Samuel I. Mann
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The authors contributed equally to all aspects of the article.

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Glossary

Rotamers

Preferred orientations of an amino acid side chain relative to the main chain.

Allostery

A biological phenomenon in which regulation occurs at a distal site often triggered by a ligand binding event, such as a metal ion.

Maquette

A simple peptide model that can be progressively altered to test the characteristics of the construction that has been commonly studied in the de novo design of proteins.

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Chalkley, M.J., Mann, S.I. & DeGrado, W.F. De novo metalloprotein design. Nat Rev Chem 6, 31–50 (2022). https://doi.org/10.1038/s41570-021-00339-5

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