Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with 
ice crystals to permit the survival of polar organisms in extremely cold 
environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium 
Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined 
to 2.1 Å resolution to gain insight into its ice-binding mechanism. The refined 
structure of FfIBP shows an intramolecular disulfide bond, and analytical 
ultracentrifugation and analytical size-exclusion chromatography show that it 
behaves as a monomer in solution. Sequence alignments and structural comparisons 
of IBPs allowed two groups of IBPs to be defined, depending on sequence 
differences between the α2 and α4 loop regions and the presence of the disulfide 
bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as 
Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) 
activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison 
of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding 
residues as well as a greater surface area in the ice-binding site. Notably, the 
ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar 
to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is 
proposed that the difference in TH activity between FfIBP and LeIBP may arise 
from the amino-acid composition of the ice-binding site, which correlates with 
differences in affinity and surface complementarity to the ice crystal. In 
conclusion, this study provides a molecular basis for understanding the 
antifreeze mechanism of FfIBP and provides new insights into the reasons for the 
higher TH activity of FfIBP compared with LeIBP.