Winter-active spiders (Clubiona) have a hyperactive antifreeze protein with a unique beta-solenoid fold.

Spiders are among the most important natural enemies of pests in orchards. Clubiona spp. are active even during winter, when other pest predators are dormant. Such activity is possible because these spiders produce antifreeze proteins (AFPs) that bind to the surface of ice crystals to prevent their growth at subzero temperatures. To investigate their AFPs, we collected Clubiona spiders from a pome fruit orchard in the Czech Republic on subzero days in December and February. The AFPs were isolated through five successive rounds of ice-affinity purification as a family of ~30-kDa isoforms. Tryptic fragments from these AFPs were sequenced using tandem mass spectrometry and corresponding transcripts were obtained from an Illumina metatranscriptome. Homologs were absent from the GenBank protein database. Protein modelling with AlphaFold2 shows that these spider AFPs form a previously unseen β-solenoid with a flat surface populated by arrays of threonine (Thr) residues. Although these features are like those of the β-solenoid AFPs from beetles and moths, the proteins are not homologous. This is a remarkable example of convergent evolution of proteins at both the structural and sequence levels for the task of binding ice.