The ice-binding site of Atlantic herring antifreeze protein corresponds to the carbohydrate-binding site of C-type lectins.
The type II antifreeze proteins (AFPs) of smelt and Atlantic herring are homologous to the carbohydrate-recognition domains (CRDs) of Ca2+-dependent (C-type) animal lectins and, like these lectins, acquire a stable and active structure upon binding Ca2+ ions. In the C-type lectin CRD, the carbohydrate-binding site is located at a Ca2+-binding site. Site-directed mutagenesis was used to test the hypothesis that the ice-binding site of the type II AFP corresponds to the carbohydrate-binding site of the lectins. To disrupt this site in the herring AFP without perturbing the Ca2+-dependent protein fold, a double mutant was constructed that changed the Ca2+- and carbohydrate-binding motif from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp to a mannose-type that has the sequence Glu-Pro-Asn and is also known to bind Ca2+. The mutant AFP exhibited proper Ca2+ binding, folding, and stability as demonstrated by ruthenium red staining, proteolysis protection assays, and CD spectroscopy. However, it showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals as does the active wild-type AFP. These results demonstrate that the ice-binding site of the herring type II AFP corresponds to the carbohydrate-binding site of the C-type lectin CRDs and further suggest that this ice-binding function evolved from the carbohydrate-binding site of a preexisting C-type lectin.
Ewart
K V
KV
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Li
Z
Z
Yang
D S
DS
Fletcher
G L
GL
Hew
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases; D005399:Fishes; D005690:Galactose / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007053:Ice; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D051381:Rats; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D021301:Antifreeze Proteins; D002352:Carrier Proteins; D006023:Glycoproteins; D007053:Ice; D037102:Lectins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C055213:saccharide-binding proteins; D010450:Endopeptidases; D002118:Calcium; D005690:Galactose