Abstract
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 de novo design of symmetric protein assemblies around metal centers with pre-defined coordination geometries. Here, we demonstrate that an artificial protein trimer termed Tet4, whose structure was designed around an idealized tetrahedral His3/H2O-ZnII coordination motif, enables the high-affinity binding of several other divalent first-row transition metal ions in the same geometry as for ZnII. We then follow the proposed evolutionary path of a natural metalloenzyme family 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.
Supplementary materials
Title
Supporting information
Description
Amino acid sequences of Tet4 variants (Table S1); crystallization conditions (Table S2); X-ray data collection and refinement statistics (Table S3); Metal binding isotherms for Tet4 (Figure S1); Structural alignment of metal-bound Tet4 (Figure S2); Distance between the N- and
C-termini of Zn-Tet4 (Figure S3); ESI-MS spectra of scTet425 variants (Figure S4); Analytical ultracentrifugation profiles of scTet4 variants (Figure S5); Michaelis-Menten plots for hydrolysis of 4MUA (Figure S6) and pNPA (Figure S8); Structural alignment of PocketTet4 (Figure S7); RFdiffusion input used to obtain the backbone of PocketTet4 (Supporting Data 1); shell script used to run ProteinMPNN to obtain the sequence of PocketTet4 (Supporting Data 2)
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