In Vivo Biogenesis of a De Novo Designed Iron–Sulfur Protein

Abstract

In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron−sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo. While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] 2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron−sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.

Catalysis and Electron Transfer in De Novo Designed Metalloproteins

“…The few reported examples of in vivo metal incorporation rely on structural motifs that permit covalent attachment of a cofactor such as c -type hemes or bilins. , Control of in vivo metal or cluster incorporation without covalent attachment remains unsolved. A recent study used the previously reported CCIS scaffold to investigate the differences between in vivo and in vitro [4Fe-4S] metalation of this construct . CCIS had been previously reconstituted in vitro using FeCl 3 , Na 2 S, and DTT under anaerobic conditions, but it showed batch to batch variability with different mixtures of oligomers …”

“…Process of producing [4Fe-4S] cluster bound CCIS in vitro vs in vivo . Reprinted with permission from ref . Copyright 2020 American Chemical Society.…”

“…(B) Structure of a three-lobed trimer contained within an antiparallel three-helix bundle to fit the observed SAXS density. Adapted with permission from ref . Copyright 2020 American Chemical Society.…”

“…This question looms over the community in part because many of these peptides are purified by reverse phase HPLC, which will strip the protein cofactors if they are not covalently linked. There have been some interesting steps in this direction, such as the work of the Anderson lab to understand the motifs necessary to incorporate c -type hemes into de novo proteins in vivo or that of Jagilinki and Nanda to investigate how in vivo incorporated [4Fe-4S] clusters differ from those incorporated in vitro . ,, Studies which investigate the metalation states of overexpressed de novo proteins are useful, but more work is needed on in vivo design strategies.…”

In Vivo Assembly of a Genetically Encoded Artificial Metalloenzyme for Hydrogen Production

“…Because metal uptake and delivery is carefully controlled by all organisms, particularly for late transition metals, incorporation of non-native metals into overexpressed proteins has remained a challenge in the field. Recent advances have demonstrated in vivo incorporation of [4Fe-4S] clusters into de novo designed peptides and assembly of c -type hemes into engineered protein scaffolds. , These approaches harness the innate biochemical machinery present in E. coli for c -type cytochrome and iron–sulfur ([FeS]) cluster maturation. As an alternative method, insertion of a complete, fully synthetic cofactor has also been shown to result in proper in vivo assembly of [FeFe] hydrogenases, which has application to generating such enzymes from an array of organisms and within diverse hosts .…”