A <i>De Novo</i> Designed Protein with VersatileMetal Binding and Tunable Hydrolytic Activity

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 <i>de novo</i> design of symmetric protein assemblies around metal centers with predefined coordination geometries. Here, we demonstrate that an artificial protein trimer, termed Tet4, whose structure was designed around an idealized tetrahedral His₃/H₂O–Zn^II coordination motif, enables the high-affinity binding of several other divalent first-row transition metal ions in the same geometry as for Zn^II. We then follow the proposed evolutionary path of a natural metalloenzyme superfamily 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.