Structure‐function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice

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Abstract
Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice‐binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66‐amino‐acid‐long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 β‐strands are paired to form 2 triple‐stranded antiparallel sheets and 1 double‐stranded anti‐parallel sheet, with the 2 triple sheets arranged as an orthogonal β‐sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154–1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface‐accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple‐stranded sheet near the C‐terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild‐type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild‐type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2‐dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C‐terminal β‐sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP‐ice interaction.