To mimic a hypothetical pathway for protein evolution, we tailor a natively monomeric protein (cytochrome cb562) for metal-mediated self-assembly, followed by re-design of the resulting oligomers for enhanced stability and metal-based functions. We show that a single hydrophobic mutation on the cytochrome cb562 surface drastically alters the outcome of metal-directed oligomerization to yield a new trimeric architecture, (TriCyt1)3. This nascent trimer coordinates metal ions using a biologically rare hexahistidine (His6) coordination site. Inspired by the example of calprotectin, a natural metal-sequestering protein that uses a His6 site to coordinate MnII with nanomolar affinity, we redesigned (TriCyt1)3 into second and third-generation variants (TriCyt2)3 and (TriCyt3)3, which feature increased structural stability and preorganization for metal coordination. The (TriCyt3)3 construct, which can trimerize in the absence of metal ions, coordinates MnII with an affinity (Kd = 50 nM) that approximates the lowest reported Kd's of the MnII-His6 site of calprotectin. While initially utilized solely for high affinity metal coordination, the TriCyt series represents a promising metallprotein scaffold for the engineering of more complex metal-based functions such as electron transfer and catalysis. We describe our efforts to modify TriCyt such that it can a) host coordinatively unsaturated metal binding sites, b) accomodate hetero-multinuclear metal coordination sites/metal clusters, and c) assemble into heterotrimeric architectures that preorganize asymmetric metal complexes. The ultimate goal of these pursuits is to design from scratch metalloproteins that can perform small molecule activation, in particular oxygen activation.

TriCyt: A de novo designed metalloprotein scaffold for pursuing complex metal-based functions

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ACS Fall 2022

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