Structure, Evolution and Engineering of Polyproline Type II Helical Antifreeze Proteins
antifreeze protein , Collembola , protein engineering , polyproline type II helix , ice-binding , protein evolution
Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including isolated and bundled alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, had not been adequately assessed. In this study, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. A range of methods was used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these collembolan AFPs had a high glycine content, a common tripeptide repeat, and were predicted to have the same polyproline type II helical bundle fold, consisting of two antiparallel layers of parallel helices, with variation in the length and number of helices. To help confirm this expectation the 9-kDa AFP from the Collembola, Granisotoma rainieri, was recombinantly expressed and its X-ray crystal structure solved to 1.21-Å resolution. Four of the nine polyproline type II helices in GrAFP form the ice-binding site supported by five helices on the other more hydrophilic side. This unique fold provided the opportunity to alter the ice-binding site area to explore how this affects antifreeze activity. A GrAFP construct with only three helices on the ice-binding site lost 90% of its activity. Whereas expansion to five helices produced a 3-fold increase in activity. The polyproline type II helical bundle AFP fold is unique to Collembola, and its prevalence within this class suggests it is ancient. Collembola arose in the Paleozoic Era with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish, which arose independently in many taxa during the Cenozoic Ice Age beginning ~30 million years ago.