The double histone fold: Structure, functional implications across the tree of life and relevance to protein design

The histone fold is one of the most ancient and versatile structural motifs in protein biology, best known for its role in chromatin organization. A remarkable variation of this motif is the double histone fold (DHF), in which two histone folds are encoded within a single polypeptide chain and assemble intramolecularly into a histone-like pseudodimer. Initially considered a rare structural anomaly, the DHF has now been identified in archaea, viruses, bacteria and eukaryotes, often in proteins unrelated to canonical chromatin architecture. In this review, we analyze the DHF from a protein engineering and evolutionary perspective, focusing on its structural determinants, modularity and repeated emergence across distant lineages. We discuss how the DHF exemplifies a robust protein design solution for DNA binding and macromolecular interaction, shaped by both divergent and convergent evolution. Finally, we highlight implications for protein design, including the DHF as a natural template for engineering DNA-binding modules that can be used, for example, to modulate chromatin organization.