Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus

Abstract

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.

Structure of a bacterial ice binding protein with two faces of interaction with ice

“…role of the cap subdomain in the overall stability of IBP [24] seem to be scaled down. This conclusion is further supported by the structure of the recently published two-domain protein IBPv [28], where each b-helix domain misses the cap subdomain but the protein has a Tm of 53.5°C [34], similar to that of capped IBP-1 fold proteins [23][24][25][26][27]. Overall, the low hydrophobicity of B and C faces might explain the only moderate TH activity of the protein.…”

“…Structural‐base sequence alignment of Efc IBP with related IBP ‐1 fold proteins. Efc IBP is aligned with Col AFP [], Ff IBP , Tis AFP 6, and Tis AFP 8 isoforms , Le I BP , and with the two domains ( dA and dB ) of IBP v . The sequence alignment has been performed using the MUSCLE program ( https://www.ebi.ac.uk/Tools/msa/muscle/) and manually corrected based on their 3D structure comparison.…”

“…Mutagenesis experiments and virtual docking to ice planes based on the crystal structures of ColAFP, FfIBP, TisAFP6, TisAFP8, LeIBP, and IBPv reveal that the IBS is located on the flattest surface of the b-helix (B face), while the other faces (A and C) are not involved in ice binding ( Fig. 2) [23][24][25][26][27][28]. These IBP-1 fold proteins typically lack regular ice-binding motifs, such as the Thr-X-Thr and the Thr-X-Asx repeats (X = any residue, Asx = Asn or Asp), typically found in insect and bacterial hyperactive IBPs [8,29].…”

“…4). Three Thr-X-Thr and one Thr-X-Asx IBS motifs are homogenously distributed along the different parallel b-strands (b2, b15, and b21) and converge to form a row of four Thr , TisAFP6, and TisAFP8 isoforms [25,26], LeIBP [27], and with the two domains (dA and dB) of IBPv [28]. The sequence alignment has been performed using the MUSCLE program (https://www.ebi.ac.uk/Tools/msa/muscle/) and manually corrected based on their 3D structure comparison.…”

An ice‐binding and tandem beta‐sandwich domain‐containing protein in Shewanella frigidimarina is a potential new type of ice adhesin

“…For instance, Do et al (2014) proposed that the greater regularity of the IBS of FfIBP relative to LeIBP explains their activity difference [24]. Similarly, Cheng et al [48] used the different amino-acid complements in the IBS of TisIBP isoforms 6 and 8 to explain their moderate-and hyperactivity, respectively. Looking at the topology of SfIBP_1's B face (the predicted ice-binding surface for all known DUF3494 IBPs), a ridge of large, bulky amino acids can be seen along the one side.…”