De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles

Abstract

De novo design provides an attractive approach to test the mechanism by which metalloproteins define the geometry and reactivity of their metal ion cofactors. While there has been considerable progress in designing proteins that bind transition metal ions including iron-sulphur clusters, the design of tetranuclear clusters with oxygen-rich environments has not been accomplished. Here, we describe the design of tetranuclear clusters, consisting of four Zn2+ and four carboxylate oxygens situated at the vertices of a distorted cube-like structure. The tetra-Zn2+ clusters are bound at a buried site within a four-helix bundle, with each helix donating a single carboxylate (Glu or Asp) and imidazole (His) ligand, as well as second- and third-shell ligands. Overall, the designed site consists of four Zn2+ and 16 polar sidechains in a fully connected hydrogen-bonded network. The designed proteins have apolar cores at the top and bottom of the bundle, which drive the assembly of the liganding residues near the center of the bundle. The steric bulk of the apolar residues surrounding the binding site was varied to determine how subtle changes in helix-helix packing affect the binding site. The crystal structures of two of four proteins synthesized were in good agreement with the overall design; both formed a distorted cuboidal site stabilized by flanking second and third-shell interactions that stabilize the primary ligands. A third structure bound a single Zn2+ in an unanticipated geometry and the fourth bound multiple Zn2+ at multiple sites at partial occupancy. The metal-binding and conformational properties of the helical bundles in solution, probed by circular dichroism spectroscopy, analytical ultracentrifugation and NMR, were consistent with the crystal structures.

Designed peptides that assemble into cross-α amyloid-like structures

“…Our discovery of a structurally well-defined cross-α structure arose from a serendipitous discovery of the packing in the crystal structure of αAm mem , an analog of the membrane-spanning Zn 2+ -transporting peptide Rocker 21 based on the backbone of a mononuclear Zn 2+ - binding four-helix bundle 22 (Supplementary Fig. 1a).…”

A general-purpose protein design framework based on mining sequence–structure relationships in known protein structures

“…Advancements introduced over the years have aimed to improve the treatment of a range of physical effects toward a more realistic representation of proteins (3,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Nevertheless, despite many examples of successful designs in the literature (18,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), it is still the case that CPD methods are not robust. State-of-the-art techniques, even in the hands of experts, fail too frequently, showing that significant inaccuracies are still present in the underlying models and motivating the development of alternative solutions.…”

Use of an Artificial Miniaturized Enzyme in Hydrogen Peroxide Detection by Chemiluminescence

“…In this respect, we developed different metal-binding sites in the context of either four-helix bundle [ 53 , 54 ] or peptide-porphyrin conjugates [ 55 , 56 , 57 ]. In the former scaffold, we afforded the design of non-heme di-iron oxidases and oxygenases, as well as the stabilization of an unprecedented tetra-zinc metal cofactor [ 58 , 59 , 60 , 61 ]. In the latter, we miniaturized and then reengineered the heme-binding site of globins, obtaining a tailor-made set of catalysts named Mimochromes (MC) [ 62 , 63 , 64 , 65 , 66 , 67 , 68 ].…”