Functional And Structural Analyses Of A Bacterial Antifreeze Protein

Abstract

Antifreeze proteins (AFPs) are a class of ice binding proteins (IBPs) that are expressed by different cold-adapted organisms to increase their freezing tolerance. AFPs have two major properties: thermal hysteresis and ice recrystallization inhibition. Here we report the functional and structural analyses of a bacterial AFP, IBPv. IBPv was originally secreted by a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica. Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2°C at concentrations higher than 50 µM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β-strands and its denaturing temperature was 53.5°C. Multiple sequence alignment of homologous IBPs predicted that IBPv contains two ice binding domains; a feature unique among known AFPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed higher ice binding activity. The 1.75 Å resolution crystal structure of IBPv was obtained, which is the first reported structure of multi-domain AFPs. The two ice binding domains are structurally similar with a RMSD of 0.68 Å by backbone alignment. Each domain consists of an irregular β-helix with a triangular cross-section and a long α-helix that parallels at one side of the β-helix. Both domains are stabilized by large numbers of tightly packed hydrophobic side chains. Structural alignment and mutagenesis studies were used to confirm a flat plane with some aligned water molecules in each domain as the ice binding site, which is located on the same face of the β-helix. Structural non-hindrance of the ice binding site was found to be pivotal for the function of IBPv. Data from thermal hysteresis and gel filtration assays suggested that the two domains could cooperate to achieve a higher ice binding effect by forming heterodimers. Direct physical linkage of the domains was required for neither the dimerization nor the cooperative effect. However, when the dimerization of the domains was disrupted by a site mutation, the cooperative effect still remained.