An intrinsically disordered domain in Polaribacter irgensii KOPRI 22228 CspB confers extraordinary freeze-tolerance

Organisms living in extremely cold environments possess mechanisms to survive low temperatures. Among the known cold-induced genes, cold-shock proteins (Csps) are the most prominent. A csp-homologous gene, cspB_Pi, has been cloned from the Arctic bacterium Polaribacter irgensii KOPRI 22228, and overexpression of this gene greatly increased the freezing tolerance of its host. This protein consists of a unique N-terminal domain and a well conserved C-terminal cold shock domain. To elucidate the detailed mechanisms involved in the extraordinary freeze-tolerance conferred by CspB_Pi, we identified the responsible domain by mutational analysis. Changes of residues in the cold shock domain that are crucial for binding RNA or single-stranded DNA did not impair the ability of the host to survive freezing stress. All domain-shuffled CspB_Pi variants containing the N-terminal domain retained the ability to confer superior freeze-tolerance. Slow electrophoretic mobility and far-UV circular dichroism spectra of the N-terminal domain suggested an intrinsically disordered structure for this region. The N-terminal domain also bound to lipid vesicles in vitro. This lipid vesicle binding characteristic is shared with other intrinsically disordered proteins, such as α-synuclein and plant dehydrins, known to confer cold-tolerance when overexpressed, suggesting a mechanism for cold-survival through membrane binding.