Anchored clathrate waters bind antifreeze proteins to ice

Abstract

The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca 2þ -bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP∶ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the "anchored clathrate" mechanism of AFP action.Ca2+ binding protein | repeats-in-toxin protein | thermal hysteresis | Antarctic bacterium | organized biohydration A ntifreeze proteins (AFPs) adsorb to the surface of ice crystals and prevent their growth (1). This adsorption lowers the freezing temperature of a solution below its melting point, enabling the survival of many organisms that inhabit ice-laden environments. Despite their common function, AFPs display remarkable diversity in their tertiary structures (2-7). This diversity results partly from their independent evolutionary origins (8, 9) and partly from the surface heterogeneity of their natural ligand, ice (10). Hexagonal ice presents many different planes (expressed as Miller indices) of water molecules to which an AFP can develop affinity. Although specificity toward different ice planes is a key determinant of antifreeze activity (11), the mechanism by which an AFP binds to ice remains undefined.Hydrogen bonds were originally proposed to be the main binding force between an AFP and ice (12). Yet this hypothesis was unable to explain how an AFP would preferentially bind ice when solvated by 55 M water. Subsequent studies proposed that the hydrophobic effect was the main ice-binding force, where constrained, clathrate-like water on the ice-binding site (IBS) is released into the solvent upon ice binding, resulting in a gain of entropy (13,14). However, several molecular dynamics (MD) simulations have indicated that the relatively hydrophobic IBS of an AFP is capable of ordering water molecules into an ice-like lattice (15-21) and, instead of shedding bound water molecules upon ice binding, the ordered waters might facilitate the AFP's interaction with ice by matching certain ice planes (15). Although intriguing, these simulations fall short of describing at the molecular level how an AFP might order water molecules into an ice-like ...

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

“…Therefore, our results show that the previous reports of AFPlike activity in dehydrins are likely due to contamination and that dehydrins do not protect enzymes by inhibiting ice growth. This also agrees with our observation that AFPs are very rigid proteins (Graether et al, 2003;Graether and Sykes, 2004) and not disordered, a property possibly required for them to form ice-like waters on their surface (Garnham et al, 2011).…”

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

“…It is now thought that the mechanism by which AFPs bind to ice is to organize ice-like waters on their IBS that merge with and freeze to the quasi-liquid layer around ice [27][28][29]. Unfortunately, the number of waters on the IBS of type III AFP in the X-ray structures is restricted by crystal contacts [30].…”

Antifreeze proteins: computer simulation studies on the mechanism of ice growth inhibition

“…48 The question naturally arises as to why hyperactive AFPs bind to multiple planes of ice crystals. Garnham et al 79 have suggested that water at the surface of ice crystals to which hyperactive AFPs are bound forms a clathrate-like structure, which can adhere to multiple planes of ice. As computer simulation is a powerful tool for analyzing the structure of water around AFPs, 80 MD simulation studies should be performed for multiple planes of ice crystals by considering the structure of water at the surface of ice crystals to which hyperactive AFP is bound.…”