Loop-Directed Cofactor Binding Enables Programmable Multicofactor Protein Design

The emergence of respiratory, photosynthetic, and assimilatory complexes in evolution required proteins capable of binding multiple catalytic and electron-transfer cofactors while exerting fine control over their spatial arrangement. Across natural systems these cofactors are preferentially positioned in loop regions. In contrast, most protein design strategies have focused on installing cofactor-binding sites within helical elements. Here we show that introducing only a pair of appropriately placed histidine ligands into the interhelical loop regions of a canonical single-chain four-helix bundle is sufficient to create new well-defined high affinity heterocofactor binding sites. This simple modification enables the self-assembly of complexes containing up to three distinct cofactors in a single designed domain with positional specificity. Using this strategy, we created constructs containing one or two hemes in combination with Zn(II)–phthalocyanine monosulfonate, Zn-heme, and the light-harvesting Zn(II)–tetraphenylporphyrin tetrasulfonate. Fluorescence measurements of constructs containing the latter show efficient energy transfer between photoactive donor cofactors. By demonstrating that loop-embedded ligands support robust, modular, and evolutionarily plausible cofactor recruitment, this work provides a mechanistic explanation for the widespread placement of redox and catalytic cofactors in loops in natural proteins: only limited packing complementarity is needed, meaning that just a few mutations can introduce a functional cofactor binding site, after which additional mutations can tune affinity, reactivity, and specificity. More importantly, it establishes a straightforward path toward constructing functional protein domains that mirror the complexity of biological energy-conversion architectures.