Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus.
Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed β-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the β-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the β-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the β-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.
Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan.
Jing
J
Cheng
Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan.
Yuichi
Y
Hanada
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Ai
A
Miura
Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Sakae
S
Tsuda
Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Hidemasa
H
Kondo
D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D012914:Snow / Q000382:microbiology*
D021301:Antifreeze Proteins; D005656:Fungal Proteins
cold adaptation; crystallography; ice growth inhibition; molecular docking; site-directed mutagenesis; thermal hysteresis