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Proc Natl Acad Sci U S A. May 3, 2011; 108(18): 7363–7367.
Published online Apr 11, 2011. doi:  10.1073/pnas.1100429108
PMCID: PMC3088597
From the Cover

Anchored clathrate waters bind antifreeze proteins to ice


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 Ca2+-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[ratio]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.

Keywords: Ca2+ binding protein, repeats-in-toxin protein, thermal hysteresis, Antarctic bacterium, organized biohydration

Antifreeze 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 (27). 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 (1521) 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 lattice that has the specificity and binding strength to irreversibly adsorb the AFP to ice.

The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large (ca. 1.5 MDa) Ca2+-dependent AFP (MpAFP) (22, 23). The protein contains two highly repetitive segments that divide it into five distinct regions (Fig. 1A). Region II of MpAFP accounts for 90% of the protein's mass and is comprised of ca. 120 tandem copies of an identical 104-aa sequence. The antifreeze activity of MpAFP resides in region IV (MpAFP_RIV), a 322-aa segment of the protein that contains 13 tandem 19-aa repeats. Recombinantly expressed MpAFP_RIV has been shown to bind Ca2+ and depress the freezing point of a solution in a hyperactive manner (23). Here, we have determined the X-ray crystal structure of MpAFP_RIV to a resolution of 1.7 Å (Table S1). The structure explains MpAFP_RIV's Ca2+-dependent hyperactivity and also provides direct experimental evidence that anchored clathrate waters bind AFPs to ice.

Fig. 1.
Structure of MpAFP_RIV. (A) MpAFP consists of five distinct regions. Highlighted in blue are the tandem 104-aa repeats that constitute region II. Region IV is colored orange. Numbers below each region indicate the number of amino acids. (B) Cartoon representation ...

Results and Discussion

MpAFP_RIV folds as a right-handed Ca2+-bound parallel beta-helix roughly 70 Å long and 20 × 10  in cross-section (Fig. 1B). Four of these beta-helices are packed within the unit cell of the crystal, each one oriented antiparallel to its two closest neighbors (Fig. 1C). Every coil of the beta-helix, typically 19 amino acids in length, contains one 6-residue Ca2+-binding turn and three short beta strands separated by Gly-rich turns (Fig. 1D). Distinct capping structures reside at the N and C termini (Fig. S1 A and B) of the beta-helix. The consensus amino acid sequence of each loop is xGTGNDxuxuGGxuxGxux, where x represents any residue (typically hydrophilic) and u residues are hydrophobic (Val, Leu, or Ile) (Fig. S1C). The u residues from each beta strand form a hydrophobic core that is screened from the solvent by main-chain hydrogen bonds that run parallel to the long axis of the helix. Thirteen Ca2+ ions align down one side of the structure and each is heptacoordinated between successive xGTGND turns (Fig. 1E). In particular, the glycines at positions 2 and 4 (xGTGND) of the upper loop contribute main-chain carbonyls whereas the aspartate at position 6 (xGTGND) contributes one side-chain carbonyl. In the lower loop, the x residue at position 1 and threonine at position 3 (xGTGND) contribute main-chain carbonyls, whereas the aspartate at position 6 (xGTGND) partially contributes both side-chain carbonyls. In this way, Asp residues at position 6 of each loop lock Ca2+ ions into place and bridge each Ca2+-binding site. This coordination scheme differs only at the most C-terminal (13th) Ca2+-binding site, where a glutamate residue extends from a recessed Ca2+-binding loop and binds Ca2+ along with two water molecules (Fig. S1D). This site is apparently the protein's most solvent-accessible Ca2+-binding site as the lanthanide Ho3+, soaked into the crystal as a heavy atom replacement for Ca2+, substituted into this position at roughly 50% occupancy in all four molecules of the unit cell. Ho3+ did not substitute at any other Ca2+-binding site.

MpAFP_RIV presents a long and flat IBS that runs the length of its Ca2+-bound side (Fig. 2A). It consists of the Thr and Asx (usually Asn) residues that project outward from the xGTGND Ca2+-binding turns. Previous site-directed mutagenesis studies have confirmed the location of the IBS (23). Glycine typically separates Thr and Asx in each turn (except in loops 6 and 8 where it is substituted by aspartate), and this pattern maintains the IBS's flatness and regularity, two characteristics that define the IBS of most AFPs, and in particular the hyperactive beta-helical insect AFPs from Tenebrio molitor (TmAFP) and spruce subworm (sbwAFP) (Fig. 2 B and C) (2, 4). Side-chain O atoms on the IBS's of all three beta-helices form rectangular arrays ca. 7.4 -  wide × 4.6 -  long (Fig. 2D) and these distances closely match the spacing of O atoms found on the primary prism and basal planes of ice. MpAFP_RIV uses a Thr-Gly-Asx motif located in a Ca2+-binding turn as its IBS, whereas both TmAFP and sbwAFP use a Thr-x-Thr motif located in a flat beta sheet to bind ice. Asx residues are required on the IBS of MpAFP_RIV to compensate for the change in beta-helical pitch that occurs in the Ca2+-binding turns of the protein, a phenomenon avoided in the beta-stranded IBSs of both TmAFP and sbwAFP. The extra length of each Asx residue allows it to align ca. 7.4 Å from a Thr of the ensuing Ca2+-binding turn. If it is remarkable that the identical IBSs of the nonhomologous beta-helical insect AFPs arose via convergent evolution, it is even more extraordinary that this precise IBS geometry has been replicated again using a distinct ice-binding motif located within a unique beta-helical fold.

Fig. 2.
IBS of MpAFP_RIV in comparison to those of TmAFP and sbwAFP. (A) The IBS of MpAFP_RIV. The Thr and Asx residues of each xGTGND Ca2+-binding turn point outward and create a flat, repetitive array ideal for ice binding. (B) The hyperactive right-handed ...

MpAFP_RIV is a homologue of the repeats-in-toxin (RTX) family of secreted virulence factors produced by numerous pathogenic Gram-negative gammaproteobacteria (24). RTX proteins are defined by the presence of a tandemly repeated nonapeptide motif (GGxGxDxux) that folds as a beta-helix with Ca2+ bound down both sides of the structure (25, 26), and not just one as seen in region IV (Fig. 3A). The xGTGND repeats of MpAFP_RIV bind Ca2+ and fold identically to the GGxGxD repeats that define the RTX proteins. However MpAFP_RIV binds Ca2+ only down one side of the helix because each 19-aa loop of the protein contains just one xGTGND Ca2+-binding repeat, and not two as is seen in the RTX proteins. Positions 3 and 5 of the canonical RTX GGxGxD Ca2+-binding turn are highly variable (Fig. 3B) and MpAFP_RIV's affinity toward ice therefore most likely developed once sufficient Thr and Asx substitutions occurred within the Ca2+-binding turns of the beta-helix.

Fig. 3.
Comparison between MpAFP_RIV and RTX-like beta-helices. (A) Cross-section of MpAFP_RIV residues 115–161 and alkaline protease (PBD ID code 1KAP) residues 331–375. Oxygens are shown in red, nitrogens blue, Ca2+ are green. Carbons are white ...

The unique IBS of MpAFP_RIV arranges water molecules into a specific ice-like lattice. These organized surface waters align down the entire length of one of the four helices in the unit cell (chain D) (Fig. 4A), and also down the bottom third of chain A (Fig. 4B). These were the only two areas in the unit cell where the IBS was completely solvent-exposed, ensuring specific protein[ratio]solvent interactions free from crystal-packing artefacts. Waters in these two separate areas are coordinated in the same manner, confirming the specificity of the interaction. The ice-like water molecules enclose the γ-methyl of each Thr (xGTGND), and this cage is anchored to the IBS by hydrogen bonds to the main-chain nitrogen and side-chain hydroxyl of each Thr (Fig. 4C). Waters are also hydrogen bonded to the main-chain nitrogen of each Gly (xGTGND) and side-chain oxygen of each Asx (xGTGND). The two Asp residues substituted for Gly (coils 6 and 8) project outward and are located in the middle of the IBS. Their side-chain carbonyls mimic the location of surface waters and also assist in their coordination (Fig. 4D). The ice-like waters that encompass the IBS of MpAFP_RIV make an excellent 3-D match to both the basal and primary prism planes of ice (Fig. 4 E and F, Fig. S2 A and B). The solvent-exposed waters located on the lower third of chain A’s IBS have an rmsd of 0.68 Å when aligned to 28 oxygen atoms on the primary prism plane of ice and 0.73 Å when aligned to 24 oxygen atoms on the basal plane of ice. This near-perfect alignment underscores the precision of the fit between the protein and ice provided by this glimpse of an AFP[ratio]ice interaction.

Fig. 4.
Ordered surface waters on the IBS of MpAFP_RIV. (A) IBS of chain D freely exposed to solvent in the unit cell. (B) Section of chain A freely exposed to solvent in the unit cell. The color scheme is the same as in Fig. 1. Ordered surface waters ...

Microscopic ice crystals formed in the presence of MpAFP_RIV are shaped as hexagonal plates (Fig. 5A), confirming the protein’s affinity toward the basal and primary prism planes. MpAFP_RIV tagged with GFP for fluorescence-based ice plane affinity (FIPA) analysis (27) turned single ice-crystal hemispheres uniformly fluorescent (Fig. 5 B and C), consistent with the protein’s affinity toward the basal and primary prism planes of ice, as well as surfaces between these planes, a property also seen with the hyperactive AFP from snow fleas (28). For a comparison, FIPA analysis was also performed on type I AFP. Type I AFP binds only the {20–21} plane of ice (Fig. 5 E and F) (29) and shapes ice into a hexagonal bipyramid with a c[ratio]a axial ratio of 3.3[ratio]1 (Fig. 5D), consistent with it binding only to this pyramidal plane and not the basal plane. MpAFP_RIV’s ability to bind to at least the primary prism and basal planes of ice accounts for its hyperactivity and further strengthens the finding (11) that basal plane binding is a key determinant of AFP hyperactivity.

Fig. 5.
Ice-binding characteristics of MpAFP_RIV and type I AFP. (A) MpAFP_RIV shapes ice into hexagonal plates at the microscopic level. The scale bar represents a length of 20 μm. (B) Single ice-crystal hemisphere of GFP-tagged MpAFP_RIV. The ...

As previously mentioned, several MD studies have reported ice-like hydration and slowed water exchange around the IBS of an AFP (1521). The structural determination of MpAFP_RIV provides a molecular explanation as to how this phenomenon might occur. The relative hydrophobicity of an AFP's IBS orders water molecules via the hydrophobic effect into an ice-like lattice that is anchored to the protein via hydrogen bonds. These anchored waters then allow an AFP to bind ice by matching a specific plane, or planes, of ice. This mechanism is applicable to any AFP regardless of activity level. Hyperactive AFPs will be able to order water molecules that allow them to adsorb to multiple planes of ice, one of which is the basal plane, while moderately active AFPs will be capable of ordering water molecules that enable them to adsorb to multiple planes of ice, just not the basal plane. The energetics that govern AFP[ratio]ice interactions have thus come full circle, with hydrogen bonds required to anchor water molecules organized by the hydrophobic effect.

Fish type III AFP was recently shown by NMR to bind ice only once the hydration shell surrounding the IBS of the protein froze, further supporting our observation (30). Though the structure of MpAFP_RIV was solved at a temperature of 100 K, it would seem reasonable to assume that at temperatures above 0 °C, the stability of anchored clathrate waters on the solvent-exposed portions of the IBS of MpAFP_RIV would decrease. In solution, waters near the IBS of an AFP are likely to be mobile and exchangeable with solvent waters. Another recent NMR study revealed that water molecules on the IBS of TmAFP displayed high mobility and exchanged with bulk water on a subnanosecond timescale (31). The short lifetime of ordered water molecules on the IBS of an AFP offers an explanation to both the concentration (11) and annealing time dependence (32) of antifreeze activity. Only a small percentage of total AFP in solution at any one time would have a quorum of water molecules in the correct orientation sufficient for ice-binding. The number of AFPs competent to bind ice would rise with increasing protein concentration and/or annealing time, increasing the amount of AFP bound to the surface of the ice crystal and therefore raising thermal hysteresis levels. In this scenario, the energetics of an AFP–ice interaction are driven primarily by enthalpic, and not entropic, gains. Shedding ordered surface waters for entropic gains would actually spoil the AFP[ratio]ice interaction, leaving the protein less able to recognize the specific plane, or planes, of ice toward which it has evolved affinity. Our results indicate that an AFP partially forms its ligand before binding to it.

Materials and Methods

MpAFP_RIV Expression and Purification.

The gene encoding MpAFP_RIV was ligated into the NdeI/XhoI sites of the pET28a expression vector placing an N-terminal 6X His-tag on the protein. The protein was expressed and purified as previously described (23). The purified protein was dialyzed against 50 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM CaCl2, and then incubated at room temperature overnight with one unit of thrombin per milligram of protein to remove the His-tag. The digestion solution was then adjusted to 5 mM imidazole and placed over an Ni-nitrilotriacetate (NTA) column (Qiagen) where the flow through was collected and dialyzed against 20 mM Tris-HCl (pH 8), 2 mM CaCl2 (buffer A).

Buffer Screen to Identify Optimal Buffer for Crystallography.

MpAFP_RIV was concentrated to 16 mg/mL in buffer A and screened via hanging drop vapor diffusion in a manner similar to ref. 33 against buffers at a concentration of 100 mM spanning a wide range of pH values (pH 3–10). Buffers with acidic to neutral pKa values caused the protein to precipitate (typically within minutes to days), while drops remained clear in wells containing alkaline buffers. A more specific buffer screen was performed using solely buffers with alkaline pKa values, while testing increasing CaCl2 concentrations in the drop (up to 10 mM). Drops containing 100 mM L-arginine (pH 9.5) remained clear at Ca2+ concentrations up to 4 mM. MpAFP_RIV was then dialyzed against 20 mM L-arginine (pH 9.5) and 4 mM CaCl2 and concentrated to 5.5 mg/mL for crystallographic screening.

MpAFP_RIV Crystallization.

Initial crystal hits were obtained via hanging drop vapor diffusion by mixing equal volumes of 5.5 mg/mL MpAFP_RIV with well solution containing 10% PEG 8000 and 200 mM MgAcetate. Crystals grew at room temperature and appeared roughly 4 wk following drop set up. The initial hit was multicrystalline in nature. Crystals suitable for structure determination were obtained using both micro- and macroseeding techniques. Briefly, the drop (4 μL) containing the initial hit was brought up to 50 μL with well solution and the crystals in the drop were crushed using a glass rod. The solution of crushed crystals was added at 1/10th volume to a drop (4 μL) containing 2.75 mg/mL MpAFP_RIV mixed with an equal volume of well solution containing 10% PEG 8000, 400 mM MgAcetate. This condition typically resulted in the growth of hundreds of small crystals (ca. 10–40-μm long). To obtain larger crystals, individual crystals were removed from the microseeded drop and successively washed for 30 to 60 s each in solutions of decreasing PEG 8000 concentration (10%, 8%, 6%) containing 400 mM MgAcetate. Crystals were then placed in a drop (4 μL) contaning 2.75 mg/mL MpAFP_RIV mixed with an equal volume of well solution containing 10% PEG 8000, 400 mM MgAcetate. Plate-like crystals (dimensions ca. 200 × 200 × 50 μm) grew within 1–2 wk. Crystals obtained following macroseeding were still multicrystalline. Placing crystals in cryo solution resulted in fracture along fault lines of individual crystal lattices, releasing single-crystal fragments (ca. 100 × 100 × 50 μm) suitable for structure determination. The cryo solution for native crystals consisted of 12% PEG 8000, 400 mM MgAcetate, and 20% glycerol. HoCl3 was used as a heavy atom for phasing and was included at a final concentration of 100 mM in an altered cryo solution consisting of 5% PEG 8000, 600 mM MgAcetate, and 30% glycerol. Crystals were allowed to soak for 5 min prior to freezing and data collection.

Structure Determination.

X-ray data were collected at 100 K at the X6A beamline (National Synchrotron Light Source). Data were integrated and scaled using HKL2000 (34). Ho3+ atoms were located using SHELX C/D/E (35) and ca. 33% of the unit cell was built automatically using ARPwARP (36). Successive rounds of model building using Coot (37) and refinement using REFMAC5 with the TLS protocol (38) were used to build the contents of the unit cell. A single protein chain from the heavy atom dataset was used as an initial search model of the native dataset using Phaser (39). The contents of the unit cell from the native dataset were built and refined in the same manner as the heavy atom dataset.

FIPA Analysis.

FIPA analysis was performed as previously described (27). DNA coding for MpAFP_RIV was ligated into the NdeI/XhoI cut sites of the pET24a vector, placing a C-terminal 6X His-tag on the protein. GFP was ligated upstream of MpAFP_RIV into the NdeI cut site of the vector. The correct clone was identified by restriction endonuclease digestion followed by DNA sequencing (Robart's Research Institute). The protein was purified via Ni-NTA chromatography and was eluted from the column in buffer containing 50 mM Tris-HCl (pH 8), 500 mM NaCl, 400 mM imidazole, and 2 mM CaCl2. The Ni-NTA eluate was dialyzed against 10 mM Tris-HCl (pH 8.0), 2 mM CaCl2, and then used for FIPA analysis. Synthetic type I AFP was labeled with tetramethylrhodamine using a previously described protocol (27). Following labeling, the protein was dialyzed against 10 mM Tris-HCl (pH 8) and then used for FIPA analysis. All hemispheres were grown at a concentration of ca. 0.1 mg/mL.

Supplementary Material

Supporting Information:


We thank Jean Jakoncic and Vivian Stojanoff of the X6A beamline at Brookhaven National Laboratories for help with X-ray data acquisition and processing and Jack Gilbert for the initial discovery of M. primoryensisAFP. We also thank Dr. John Allingham for access to a Rigaku home X-ray source for initial diffraction experiments. This work was funded by the Canadian Institutes of Health Research. C.P.G. is the recipient of an Natural Sciences and Engineering Research Council of Canada three-year Postgraduate Scholarship (PGS D3). P.L.D. holds a Canada Research Chair in protein engineering.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1100429108/-/DCSupplemental.

Data deposition: The atomic coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.rcsb.org (RCSB PDB ID code 3P4G).

See Commentary on page 7281.


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