Design of functional metalloproteins

Abstract

Metalloproteins catalyse some of the most complex and important processes in nature, such as photosynthesis and water oxidation. An ultimate test of our knowledge of how metalloproteins work is to design new metalloproteins. Doing so not only can reveal hidden structural features that may be missing from studies of native metalloproteins and their variants, but also can result in new metalloenzymes for biotechnological and pharmaceutical applications. Although it is much more challenging to design metalloproteins than non-metalloproteins, much progress has been made in this area, particularly in functional design, owing to recent advances in areas such as computational and structural biology.

Figure 1. Designed metalloproteins using de novo designed scaffolds

a, Computer model of a bis-His ligated mono-heme α-helical bundle. Adapted from ref courtesy of William DeGrado. b, Computer model of a bis-His ligated multi-heme four α-helical bundle. Reproduced from ref. c, Computer model of Zn(II) bound His₂Cys₂ motif of a Zn-finger mimic. Reproduced from ref. d, X-ray crystal structure of As(III) bound three-stranded coiled-coil (PDB 2JGO). e, X-ray crystal structure of di-Zn(II) Due Ferro 1; PDB 1EC5).

Figure 2. Designed metalloproteins using native scaffolds

a, Native nickel binding site of NikR (left) and the re-engineered UO₂²⁺ binding site of the mutated NikR (right). b, Loop directed mutagenesis of blue copper azurin to yield the dinuclear, purple Cu_A azurin construct. c, Catalytic heme-copper center in CcO (left) and the designed heme-cooper model in sperm whale Mb (right).

Figure 3. Site specific incorporation of unnatural amino acids into a protein scaffold for tuning of metal properties

Crystal structures of a, rubredoxin (PDB: 1CAA) and b, azurin (PDB: 4AZU) showing variant residue location for unnatural amino acid incorporation.

Figure 4. Strategies for non-native cofactor incorporation into a protein scaffold

Native cofactor substitution of heme b in myoglobin for a, Fe-porphycene and b, 3,3-Cr salophen, exploiting the structural and dative-bonding similarities between the native cofactor and the surrogate. Affinity tagging of the catalyst with a protein-selective linker such as the biotin-streptavidin couple, c, allows for versatile attachment of non-native cofactors. Covalent strategies include a single attachment strategy as in d, showing the adipocyte lipid binding protein (ALBP) linked through Cys117 to a phenanthrolene complex; and dual covalent attachment as in e, showing dual anchoring of a Mn-salen complex into the myoglobin scaffold. Description of streptavidin complex obtained from ref. Crystal structure of Fe porphycene, Cr(3,3′-Me₂-salophen)-Mb complex, biotin-streptavidin complex, and phenanthroline-ALBP were obtained from the Protein Databank (PDB codes: 2D6C, 1J3F, 2QCB, and 1A18, respectively). Computer model of Mb(L72C/Y103C) generated from an overlay with native heme b structure.

Figure 5. Close match between a designed metalloprotein and its native target protein

Crystal structures of a designed Cu_A site in azurin (PDB code 1CC3) and a Cu_A site in native CcO (PDB code: 1AR1) as viewed from above the Cu₂(S_cys)₂ plane (a), and along the Cu₂(S_cys)₂ plane.