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 ²⁺ -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.

Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.

Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80 kDa large subunit and a common 28 kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure–function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350 μM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.

The crystal structure of an antifreeze protein shows a polypentagonal network of water in the protein core. [Also see Perspective by Sharp ]

AFPs (antifreeze proteins) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to, and inhibiting, the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large(>1 MDa) hyperactive Ca2+-dependent AFP. We have cloned,expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized that shows similarity to the RTX (repeats-in-toxin) family of proteins. The recombinant protein requires Ca2+ for structure and activity, and it is capable of depressing the freezing point of a solution in excess of 2 degrees C at a concentration of 0.5 mg/ml, therefore classifying it as a hyperactive AFP. We have developed a homology-guided model of the antifreeze region based partly on the Ca2+-bound beta-roll from alkaline protease. The model has identified both a novel beta-helical fold and an ice-binding site. The interior of the beta-helix contains a single row of bound Ca2+ ions down one side of the structure and a hydrophobic core down the opposite side. The ice binding surface consists of parallel repetitive arrays of threonine and aspartic acid/asparagine residues located down the Ca2+-bound side of the structure. The model was tested and validated by site-directed mutagenesis. It explains the Ca2+-dependency of the region, as well its hyperactive antifreeze activity. This is the first bacterial AFP to be structurally characterized and is one of only five hyperactive AFPs identified to date.AFPS