Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins

Y Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

Automated summary

By studying different isoforms of III antifreeze proteins, it was found that the QAE1 isoform exhibited the highest activity, while mutant constructs of the QAE2 and SP isoforms displayed similar thermal hysteresis and ice recrystallization inhibition activities compared to the QAE1 isoform. In terms of cryopreservation, the QAE1 isoform and active mutants showed better preservation of follicle morphology and more efficient prevention of DNA double-strand breaks. Additionally, active AFPs formed unique hydrogen bonds with the first 310 helix, playing a crucial role in the efficient binding to ice crystals, which was disrupted in inactive isoforms.