Antifreeze proteins from spruce budworm (Choristoneura fumiferana): Crystal structures and functional insights

Antifreeze proteins (AFPs) have been identified in a number of organisms that must survive sub-zero temperatures. These proteins adsorb to seed ice crystals and inhibit their growth, providing freeze-protection. Primary sequences and three-dimensional structures of characterized AFPs are highly varied and therefore bring forth a compelling structure-function question: how do these diverse proteins bind to ice and inhibit its growth?; The insect spruce budworm (Choristoneura fumiferana) (Cf) has a number of isoforms of its potent AFP. In order to understand how these AFPs elicit their thermal hysteresis activity, this study focuses on two CfAFP isoforms that are 66% identical in terms of protein sequence and vary in their molecular weight, 9 kDa (CfAFP-337) and 12 kDa (CfAFP-501), respectively.; Recombinant CfAFP-337 was crystallized, employing a high-temperature incubation technique, and the 2.3 Å resolution X-ray crystallographic structure of CfAFP-337 was solved using iodide-based single anomalous scattering. The structure reveals a highly regular left-handed β-helical platform with the ice-binding face of the protein forming an ordered two-dimensional Thr array. This structure enabled a model of CfAFP-ice-docking to be developed, based on a surface complementarity model, which may prove to be generally applicable to both fish and insect AFPs.; The higher molecular weight recombinant CfAFP-501 isoform was produced in Escherichia coli and purified. The antifreeze activity of the protein was augmented ∼3-fold as compared to CfAFP-337. The structure of CfAFP-501 was resolved by molecular replacement, using a theoretical probing model, and confirmed that the 31 additional amino acids form two extra β-helical loops, essentially extending the ice-binding surface of the protein. A deletion mutant of this protein was made (CfAFP-501-Δ-2-loop) and found to have activity similar to that of CfAFP-337, demonstrating that the size of CfAFP-501 is the key determinant of its higher activity.; This study contributes to the debate involving how AFPs bind to ice. Using the crystal structures of two CfAFP isoforms an ice-binding model based on protein-ice surface complementarity is proposed. In addition, the study reveals a compelling link between the size of a CfAFP isoform and its thermal hysteresis activity.