Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein ( AFP) to assist in the protection of the overwintering larval stage. AFPs ar e thought to lower the freezing point of the hemolymph, noncolligatively, b y interaction with the surface of ice crystals. previously we had identifie d a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activ ity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperacti vity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isol ated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes acco rding to the length of their 3' untranslated regions (UTRs). The new isofor ms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a resul t of a 30 amino-acid insertion bearing two additional ice-binding motifs sp aced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional P-hel ix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs an often substituted by Val, IIe or Arg and a r ecombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AEP isoforms s uggests therefore that specific substitutions at the first Thr in the ice b inding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely i mportant for the dynamics of initial ice contact and interaction by these h yperactive antifreezes.