Abstract Homochirality of biopolymers emerged early in the history of life on Earth, nearly 4 billion years ago. Whether the establishment of homochirality was the result of abiotic physical and chemical processes, or biological selection, remains unknown. However, given that significant events in protein evolution predate the last universal common ancestor, the history of homochirality may have been written into some of the oldest protein folds. To test this hypothesis, the evolutionary trajectory of the ancient and ubiquitous helix-hairpin-helix (HhH) protein family was analyzed for functional robustness to total chiral inversion of just one binding partner. Against expectations, functional ‘ambidexterity’ was observed across the entire trajectory, from phase separation of HhH peptides with RNA to dsDNA binding of the duplicated (HhH) 2 -Fold. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed significant overlap between the binding modes of a natural and a mirror-image protein to natural dsDNA. These data suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed, and that the HhH protein family is an intriguing exception to the dogma of reciprocal chiral substrate specificity proposed by Milton and Kent (Milton et al . Science 1992).
