Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein 
(LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point 
below the melting point once it binds to ice. LeIBP is a member of a large class 
of ice-binding proteins, the structures of which are unknown. Here, we report 
the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- 
and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed 
a dimeric right-handed β-helix fold, which is composed of three parts: a large 
coiled structural domain, a long helix region (residues 96-115 form a long 
α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic 
loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop 
region has an extended conformation pointing away from the body of the coiled 
structural domain and forms intertwined dimer interactions. In addition, 
structural analysis of glycosylated LeIBP with sugar moieties attached to 
Asn(185) provides a basis for interpreting previous biochemical analyses as well 
as the increased stability and secretion of glycosylated LeIBP. We also 
determined that the aligned Thr/Ser/Ala residues are critical for ice binding 
within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a 
common β-helical fold similar to that of canonical hyperactive antifreeze 
proteins, the ice-binding site is more complex and does not have a simple 
ice-binding motif. In conclusion, we could identify the ice-binding site of 
LeIBP and discuss differences in the ice-binding modes compared with other known 
antifreeze proteins and ice-binding proteins.