Metals play an important role in human health with functions in enzyme catalysis, bone health, cellular respiration, and immune function. However, too much metal or the wrong type of metal can be poisonous to our cells by causing DNA damage or disruption of normal functions, or simply by overwhelming our organs’ ability to detoxify them. Researchers have developed models designed to study the details of protein-metal interactions with the hope that increased understanding of these complex systems may facilitate the development of enzymes with new catalytic abilities or peptides that can detoxify heavy metal exposures. A recent report of work conducted at the U.S. Department of Energy’s Advanced Photon Source (APS) and published in the journal Nature Chemistry highlights one research group’s big step toward de novo enzyme design. This work paves the way for the design of novel catalytic and metal-binding peptides with unique properties for a variety of applications in heavy metal biochemistry.
The work was part of a collaboration between researchers at the University of Michigan, the University of Miami, and King Mongkut’s University of Technology (Thailand). Previous work from this group had shown that short peptides consisting of 4 or 5 7-amino acid repeats could be designed to form three-stranded coiled-coil scaffolds that could bind a metal ion. However, although the scaffold oriented the metal ion near a catalytic histidine, as is observed in many enzyme active sites, the symmetry in the three-stranded coil inhibited critical chemical interactions. The designed enzyme performed well but was still 300-fold less efficient than the enzymes they were modeling. The group hypothesized that if they could generate coiled coils using two different types of peptides rather than three of the same peptide, they might be able to make an asymmetric site in their enzyme for the needed chemistry to occur.
The group started by designing peptides based on the previously successful version but with some changes that would hopefully bias the three-stranded coiled-coil structures to fold with two different types of peptides. The main design for the peptides was to have the same basic 7-amino acid sequence for 4 or 5 repeats but vary a hydrophobic amino acid at positions 1 and 4 to a metal-binding amino acid at critical locations. For example, in the 7-amino acid sequence, two paired peptides would have the same basic sequence, including a cysteine amino acid to bind to a lead ion, but one would have a leucine at the fourth position while the other would have an alanine at that position, creating a little more space in the coil for differential packing of water molecules. The team also varied whether one or both peptides would have a hydrogen bonding residue in addition to the catalytic histidine at a critical location. After developing over a dozen versions of the peptides, the group set about testing which combinations would form mixed coiled-coil structures and whether they had the correct orientation of all of the important catalytic players.
The first step was to mix the peptides and confirm that they formed the correct three-stranded coiled-coil structures. The team used x-ray crystallography at the Life Sciences Collaborative Access Team 21-ID-F x-ray beamline at the APS to confirm that they could indeed form three-stranded coiled coils (Fig. 1). (The APS is an Office of Science user facility at Argonne National Laboratory.) Quantum mechanics/molecular mechanics analyses identified two versions that had the most favorable complexation energies, forming the most stable structures. In order to be sure that they had a homogeneous solution of only one type of coil (for example, A2B only and not A2B plus A3 and B3) they performed 207Pb nuclear magnetic resonance spectroscopy on the samples. The samples generated one peak, suggesting that the sample was purely one type of coil. Finally, in an important first step towards testing the ability of their three-stranded coils to mimic enzymatic catalysis, the team was able to bind zinc to the catalytic histidine without disrupting the coiled structure.
These results show that it is possible to design three-stranded coiled-coil protein structures that can mimic the metal-binding active sites of known enzymes using a combination of two different types of peptides, providing for variability in functional uses. ― Sandy Field
See: Audrey E. Tolbert 1, Catherine S. Ervin1, Leela Ruckthong2, Thomas J. Paul3, Vindi M. Jayasinghe-Arachchige3, Kosh P. Neupane1, Jeanne A. Stuckey1, Rajeev Prabhakar3, and Vincent L. Pecoraro1, “Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)3 template mediated strategy,” Nat. Chem. 12, 405 (April 2020). DOI: 10.1038/s41557-020-0423-6
Author affiliations: 1University of Michigan, 2King Mongkut’s University of Technology, 3University of Miami
Correspondence: * vlpec@umich.edu
The authors acknowledge funding from National Institutes of Health grant no. R01 ES012236, National Science Foundation grant no. CHE-1664926, and the Skill Development Grant from King Mongkut’s University of Technology, Thonburi, Thailand. Use of the Life Sciences Collaborative Access Team x-ray facility was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817).
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
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