Poster Presentation 50th Lorne Proteins Conference 2025

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain (#210)

Orit Dr Ktorza Weil 1 , Segev Mr Naveh Tassa 2 , Yael Dr Fridmann Sirkis 2 , Kesava-Phaneendra Dr Cherukuri 2 , Dragana Dr Despotović 3 , Norman Dr Metanis 1 , Yaacov Professor Levy 2 , Liam Professor Longo 4
  1. Institute of Chemistry, The Center for Nanoscience and Nanotechnology, Casali Center of Applied Chemistr, The Hebrew University of Jerusalem, Israel, Jerusalem, Israel
  2. Weizmann Institute of Science, Rehovot, Israel
  3. Institute of Molecular Genetics and Genetic Engineering , University of Belgrade , Belgrade, Serbia
  4. Earth-Life Science Institute , Tokyo Institute of Technology , Tokyo, Japan

The helix-hairpin-helix (HhH) motif is an ancient and ubiquitous nucleic acid-binding element that has emerged as a model system for studying the evolution of dsDNA-binding domains from simple peptides that phase separate with RNA. We analyzed the entire putative evolutionary trajectory of the HhH motif – from a flexible peptide to a folded domain – for functional robustness to total chiral inversion. Against expectations, functional ‘ambidexterity’ was observed for both phase separation of HhH peptides with RNA and binding of the duplicated (HhH)2-Fold to dsDNA. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed overlap between the binding modes adopted by the natural and mirror-image proteins to natural dsDNA. The retention of several dissociation phases upon chiral inversion may reflect the history of (HhH)2-Fold binding, with the ultimate emergence of a high-affinity binding mode depopulating but not displacing more primitive (ambidextrous) modes. These data underscore the surprising functional robustness of the HhH protein family and could suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed. 

  1. bioRxiv 2023.03.06.531422; doi: https://doi.org/10.1101/2023.03.06.531422