Recurrent catalytic-site change and electrostatic remodeling across the HEPN ribonuclease fold

When evolution removes a residue essential for catalysis, the protein fold may survive, but whether the former active site simply decays or is physicochemically remodeled remains unclear. We explored this question in the HEPN family of ribonucleases. HEPN proteins normally cleave RNA using a conserved R-X(4-6)-H motif, in which histidine plays a central catalytic role. Using the structure-first workflow now implemented as FOLDTRACE, we searched the AlphaFold/UniProt50 representative database for HEPN-fold proteins without requiring retention of the catalytic motif. Among the 1,000 top-ranked hits, 836 lacked an explicit HEPN name or attached HEPN-clan Pfam cross-reference. Of these, 750 were DUF86/UPF0331 proteins already recognizable as HEPN-related at the family level, illustrating incomplete per-record cross-referencing rather than discovery of an unknown HEPN family. Eighty-six proteins lacked the canonical catalytic histidine, and most retained the ancestral HEPN cleft. Phylogenetic analyses showed repeated H/non-H state changes. Monomer-level electrostatic screening identified a subset with strong negative shifts, but dimer reconstruction showed why these calls require oligomeric validation: A0A852UQU6 retained a strong shift at the composite active site, whereas another monomer-level candidate did not. Together, these results establish recurrent catalytic-state change and physicochemical remodeling of the ancestral HEPN cleft. Whether such remodeling corresponds to altered binding, regulation, or another molecular function remains a hypothesis requiring experimental testing.