Highly conserved, positively charged tandem repeats mediate multimerization of ice nucleation proteins.

Ice nucleating bacteria like Pseudomonas borealis ( Pb ) can trigger frost formation at temperatures as high as -2 °C. The efficiency of ice formation by these bacteria comes from the ability of their >100 kDa ice nucleation proteins (INPs) to form ∼10 MDa multimers. The INP monomers contain an array of tandem repeats, each of which is predicted to form a single coil in a β-solenoid. The 12 coils at the C-terminal end of the INP differ from the other 53 coils in lacking water organizing motifs and by having arginines on one side of the solenoid in place of acidic residues. Here we show that these arginine-containing coils (R-coils) are a distinct subdomain that, along with the adjacent putative 41-residue capping structure, facilitate INP multimerization. Bioinformatic analyses show that the cap structure is highly conserved, as is the number of R-coils. Indeed, the loss of just a few R-coils eliminated ice-nucleation activity, as did mutations designed to spoil the fold of the capping structure. Movement of the R-coils to the N-terminal end or to the middle of the β-solenoid caused a large decrease in ice nucleation temperature. Replacing the arginines with acidic residues decreased the nucleation temperature. Activity was restored when these residues were in turn replaced by lysines. A role for electrostatic interactions in INP self-assembly was suggested by the effect of pH on ice nucleation activity in bacterial lysates. This activity declined by 3-6 °C at pH values below 5.0. When the INP-producing E. coli were examined by cryo-electron tomography, the cell cytoplasm contained clusters of 100 to 200 nm-long and ∼5 nm-wide fibres that were absent from control bacteria. Each fibre is wide enough to have up to two INPs in cross-section and is long enough to span several INPs end to end. The ice nucleation activity in the lysates was remarkably resistant to heat treatment up to ∼70 °C, above which there was a slight decline with increasing temperature. Even after heating to 99 °C, the lysate ice nucleation activity only lost ∼8°C. We suggest that the R-coils play a crucial role in INP fibre assembly and that a bundle of these fibers could amass a sufficient number of ice-like water molecules to initiate ice nucleation at high sub-zero temperatures.