The ice-binding site of sea raven antifreeze protein is distinct from the carbohydrate-binding site of the homologous C-type lectin

Antifreeze proteins lower the freezing point of their solution by binding t o ice and inhibiting its growth. One of several structurally different anti freeze proteins in fishes (type II) is homologous to the carbohydrate-recog nition domain of Ca2+-dependent lectins and adopts the same three-dimension al fold. Type II antifreeze proteins from herring and smelt require Ca2+ fo r binding to ice, whereas this same antifreeze protein in sea raven binds t o ice in the absence of Ca2+ and has only two of the five Ca2+-liganding am ino acids that are present in the lectin. To locate the ice-binding site, s ite-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven an tifreeze protein were produced by secretion from Pichia pastoris. Pairs of amino acid replacements, insertions, and a peptide loop swap were made in t he region equivalent to the sugar-binding site of the lectin that encompass es loops 3 and 4 and beta-sheets 7 and 8. Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca2+-binding site are only peripherally invo lved in ice binding, When adjacent surface residues were mutated, the repla cement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation, This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corres ponding to the proposed CaCO3-binding surface of a third homologue, the pan creatic stone protein. Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein ge ne than to the lectin gene. These results support the notion that this fami ly of proteins has evolved more than one binding surface from the same prot ein scaffold.