Crystal structure and nucleic acid binding activity of the conserved UPF0235 family protein PF1765 from Pyrococcus furiosus.

The UPF0235 UniProt family proteins are conserved across archaea, bacteria, and eukaryotes; however, they remain functionally uncharacterized. Here, we report the high resolution (1.3 Å) crystal structure of UPF0235 protein (PF1765, UniProt: Q8U052) from Pyrococcus furiosus. The structure exhibits a compact α+β fold with a β(4)-α-β(2)-α topology, forming a pseudo β-barrel comprising predominantly antiparallel six β-strands encasing two α-helices. Structural comparisons via DALI revealed no significant similarity to known functional proteins, indicating PF1765's unique topology. Despite the low sequence identity (20-35 %), AlphaFold3 models of PF1765 homologs exhibit high structural conservation (RMSD <1.63 Å) across all domains of life, suggesting strong evolutionary pressure to preserve its 3-D structure. The biophysical characterization of PF1765 confirms the monomeric nature and thermal stability up to 95 °C, consistent with its hyperthermophilic origin. In silico predictions of nucleic acid binding potential of PF1765 was substantiated by biochemical evidence using electrophoretic mobility shift assays and fluorescence polarization experiments. We demonstrated that PF1765 binds variably to dsDNA, ssDNA, and RNA, despite lacking canonical nucleic acid binding domains. Our study provides the first structural and functional characterization of a member of the UPF0235 protein family, revealing PF1765 as a thermostable, evolutionarily conserved, and structurally unique nucleic acid-binding protein.