Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice

FEBS J. 2021 Jul;288(14):4332-4347. doi: 10.1111/febs.15717. Epub 2021 Feb 3.


A springtail (Collembola) identified as Granisotoma rainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~ 6.9 and 9.6 kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21 Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastrura harveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

Keywords: Collembola; X-ray crystallography; antifreeze protein; bound waters; ice lattice.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antifreeze Proteins / chemistry*
  • Arthropod Proteins / chemistry*
  • Arthropods / metabolism*
  • Crystallography, X-Ray
  • Hydrogen Bonding
  • Hydrophobic and Hydrophilic Interactions
  • Ice*
  • Molecular Dynamics Simulation
  • Peptides / chemistry*
  • Water / chemistry*


  • Antifreeze Proteins
  • Arthropod Proteins
  • Ice
  • Peptides
  • Water
  • polyproline

Associated data

  • GENBANK/MT780727
  • GENBANK/MT780728
  • GENBANK/MT780729
  • GENBANK/MT780730
  • GENBANK/MT780731
  • GENBANK/MT985982