Variations on a theme: non-canonical DUF3494 ice-binding proteins.

Among the many ice-binding proteins (IBPs) found in microorganisms (bacteria, archaea, fungi and algae), the canonical DUF3494 beta-barrel type is the most common. Until now, little variation has been found in this structure: an initial coil leads into an alpha helix that directs the following coils into a reverse stack, with the final coil ending up next to the initial coil. Here, I show that there exist many bacterial proteins whose AlphaFold-predicted structures deviate from the DUF3494 structure so that they are not recognized as belonging to an existing DUF or Pfam family. In these non-canonical DUF3494 (ncDUF3494) proteins, the number of coils in the alpha helix is highly variable, often being as high as 14. The putative ice-binding sides of each of 13 proteins modeled have a well-aligned row of hydrophilic residues, with spacings that are close to the repeat distance on the ice a-axis. A recombinant protein made for one of the proteins showed that it had ice-binding activity, even in the µg/ml range. The ncDUF3494 proteins appear to be found only in bacteria, the great majority of which live in icy habitats. C-terminal PEP-Cterm motifs, which are rare in DUF3494s, are present in most of the ncDUF3494s, possibly indicating a secretory function. The relatively narrow distribution of ncDUF3494 proteins suggests that they are a later development in DUF3494 evolution.