Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Ice nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Here we have predicted a novel β-helical fold for the INP produced by the bacterium <jats:italic>Pseudomonas borealis</jats:italic>. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which <jats:italic>Pb</jats:italic> INP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the <jats:italic>Pb</jats:italic> INP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As <jats:italic>Pb</jats:italic> INP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.</jats:p> </jats:sec>