Journal of Molecular Biology
Antifreeze Protein from Freeze-Tolerant Grass Has a Beta-Roll Fold with an Irregularly Structured Ice-Binding Site
Graphical Abstract
Highlights
► Some ice-binding (antifreeze) proteins (IBPs) help organisms tolerate freezing. ► The first structure of a plant IBP has been solved by X-ray crystallography. ► IBP from grass has a novel beta-roll fold with eight similar coils. ► The IBP's IBS is irregular compared to most AFPs. ► An irregular IBS may favor inhibition of ice recrystallization.
Introduction
Antifreeze proteins (AFPs) are found in diverse organisms ranging from vertebrates to bacteria that are exposed to sub-freezing environments at some point in their life histories.1, 2, 3, 4 Two frequently measured activities of AFPs are depression of the freezing point relative to the melting point, referred to as thermal hysteresis (TH),5 and ice recrystallization inhibition (IRI).6 TH activity halts ice crystal growth, allowing freeze-intolerant organisms (such as fish and some insects) to supercool and avoid freezing. IRI activity is more relevant in freeze-tolerant organisms, which are unable to avoid freezing and therefore must minimize the damage caused by recrystallization of ice in frozen tissue.7 For example, some overwintering plants such as carrots and grasses produce proteins that show weak TH activity but have IRI activity matching or exceeding that of fish and insect AFPs.8, 9, 10 The structural basis for the differences in these activities is one of the issues addressed in this work. Because some proteins with TH activity function as IRI proteins rather than antifreezes, we introduce the more inclusive term “ice-binding protein” (IBP) to refer to proteins that serve in either capacity.11
IBPs irreversibly adsorb to the surface of ice crystals and inhibit their growth by the Gibbs–Thomson effect,12 in which the addition of water molecules to the ice lattice is limited to the space between bound IBPs, resulting in surface curvature that makes the further addition of water thermodynamically unfavorable.13 Recent work suggests that IBPs adsorb to ice through an ordered ice-like array of waters assembled by the protein's ice-binding site (IBS), which merges with and freezes to the ice surface.14, 15, 16 The spacing of these so-called “clathrate” waters, anchored to the IBP by hydrogen bonds, matches one or more planes of ice. In this anchored-clathrate mechanism, the IBP essentially preforms part of its ligand before irreversibly adhering to it.
AFPs were recognized in certain insects over 30 years ago.17, 18, 19 More recently, characterization of purified insect AFPs revealed that their specific TH activities exceed those of fish AFPs by an order of magnitude;20, 21 thus, they were termed “hyperactive.” The heightened TH of hyperactive versus moderately active AFPs has been attributed to their unique affinity for the basal plane of ice.22 Moderately active AFPs bind only to prism and/or pyramidal planes, leaving the basal planes unprotected and vulnerable to growth.23, 24, 25, 26, 27 Although the addition of water to the basal planes of ice is less favorable than to a prism surface, this readily occurs under supercooled conditions when the TH gap of moderately active AFPs is exceeded, resulting in rapid crystal growth along the c-axis. Adsorption of AFPs to the basal plane provides substantially better coverage of the ice crystal and therefore provides greater TH activity.
The perennial ryegrass Lolium perenne is a freeze-tolerant forage grass planted worldwide28 that thrives in cold environments.29, 30 An IBP isolated from L. perenne (LpIBP) was reported to have high IRI but low TH activity.8 LpIBP TH activity is an order of magnitude lower than that of hyperactive AFPs and slightly less than that of moderately active fish type I, II and III AFPs.31 However, it has superior IRI activity.8, 10 Subsequently, several isoforms and homologues of this IBP have been found in various grasses in the sub-family Pooideae.32 The protein originally isolated constitutes the highly repetitive C-terminal “IRI” domain of a longer protein. On the basis of the DNA sequence, the complete protein includes an N-terminal secretion signal sequence and a leucine-rich repeat domain, but the ice-binding activity is limited to the IRI domain, which may represent the fully processed form of the protein. LpIBP is secreted from plant cells and functions in the apoplast, where it acts to minimize frost damage to the plant tissue, largely through its IRI activity.28
The sequence of the highly repetitive C-terminal domain of LpIBP suggested that its structure might resemble other beta-solenoid proteins, in particular the hyperactive AFPs discovered in the insects Tenebrio molitor (TmAFP) and Choristoneura fumiferana (CfAFP).33, 34 This was supported by a computer model of this protein as a right-handed beta-roll with two flat beta-sheets on opposite sides of the solenoid termed “a” and “b” faces.35 These flat surfaces resembled the IBSs of the insect AFPs, and it was originally proposed that both sheets might simultaneously bind ice on opposite sides of LpIBP. However, it was subsequently determined experimentally that only one of the two flat surfaces is responsible for TH activity.31 The introduction of single steric point mutations on the a face of LpIBP reduced the TH and IRI activities of the protein by up to 90%, whereas mutation of the b face had only a small effect.
Here, we report the X-ray crystal structure of LpIBP, which is the first such description of a plant IBP. Its structure resembles that of some of the hyperactive AFPs but with a less regular IBS, which we argue is the basis for its low TH activity. Like the hyperactive AFPs, LpIBP binds the basal plane of ice. Together, these analyses suggest that LpIBP has developed the ability to control ice growth at high sub-zero temperatures, which is compatible with a role in promoting freeze tolerance.
Section snippets
Crystallization and structure determination of LpIBP
LpIBP crystallized in the monoclinic C2 space group with two molecules per asymmetric unit. Crystals were grown at low pH (4.2) and high concentrations of ethanol (40–50%) at 4 °C over approximately 4 weeks. Data were collected at beamline X6A at National Synchrotron Light Source in Brookhaven National Laboratories. Two data sets obtained from two crystals were combined to obtain phases using single isomorphous replacement with anomalous scattering. After solvent flattening, the electron
Discussion
Here, we have presented the first structure of a plant “antifreeze protein,” which is in effect the first structure of an IBP that functions primarily in freeze tolerance by inhibiting ice recrystallization. The fish and insect AFP structures solved to date25, 33, 34, 42, 43, 44, 45, 46 all come from organisms that must avoid freezing. The repetitive, beta-helical LpIBP structure provides a flat regular ice-binding face that resembles several AFPs, most notably the hyperactive AFPs from
Purification and crystallization of LpIBP
Recombinant LpIBP with a C-terminal 6 × His-tag was purified in three steps as previously described.31 After removal of most contaminating Escherichia coli proteins by boiling, LpIBP was further purified by ice affinity purification,65 which also selected for properly folded LpIBP. Finally, Ni-Agarose affinity chromatography was performed, mainly to concentrate the His-tagged product. The pure protein was eluted and dialyzed against 50 mM Tris–HCl (pH 7.8), 100 mM NaCl and 1 mM
Acknowledgements
This research was funded by a grant to P.L.D. from the Canadian Institutes of Health Research. The authors thank Debborah Fass for the gift of GFP-TmAFP, Christopher Garnham for help growing ice crystal hemispheres, Jean Jakoncic and Vivian Stojanoff from beamline X6A at Brookhaven National Laboratory for beamline support, Margaret Daley and Brian Sykes for validating the folding of the TmAFP point mutations, Sherry Gauthier for technical support, Zongchao Jia and John Allingham for access to
References (73)
- et al.
Antifreeze proteins: an unusual receptor–ligand interaction
Trends Biochem. Sci.
(2002) - et al.
Plant thermal hysteresis proteins
Biochim. Biophys. Acta
(1992) - et al.
Antifreeze proteins in overwintering plants: a tale of two activities
Trends Plant Sci.
(2004) - et al.
Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis
Cryobiology
(2010) - et al.
Isolation and characterization of freezing-point depressing peptides from larvae of Tenebrio molitor
Comp. Biochem. Physiol. B Comp. Biochem.
(1980) - et al.
The basis for hyperactivity of antifreeze proteins
Cryobiology
(2006) - et al.
Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes
Biophys. J.
(1991) - et al.
Understanding the mechanism of ice binding by type III antifreeze proteins
J. Mol. Biol.
(2001) - et al.
Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity
Biophys. J.
(2008) - et al.
Molecular mechanisms underlying frost tolerance in perennial grasses adapted to cold climates
Plant Sci.
(2011)
How do plants survive ice
Ann. Bot.
Identification of the ice-binding face of a plant antifreeze protein
FEBS Lett.
A theoretical model of a plant antifreeze protein from Lolium perenne
Biophys. J.
Beta-rolls, beta-helices, and other beta-solenoid proteins
Adv. Protein Chem.
Crystallographic insights into the autocatalytic assembly mechanism of a bacteriophage tail spike
Mol. Cell
Structural modeling of snow flea antifreeze protein
Biophys. J.
Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne
Cryobiology
Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne
Cryobiology
Identification of the ice-binding face of antifreeze protein from Tenebrio molitor
FEBS Lett.
The effects of steric mutations on the structure of type III antifreeze protein and its interaction with ice
J. Mol. Biol.
New ice-binding face for type I antifreeze protein
FEBS Lett.
Valine substituted winter flounder ‘antifreeze’: preservation of ice growth hysteresis
FEBS Lett.
Structure–function relationships in a type I antifreeze polypeptide. The role of threonine methyl and hydroxyl groups in antifreeze activity
J. Biol. Chem.
Purification of antifreeze proteins by adsorption to ice
Biochem. Biophys. Res. Commun.
Rapid and efficient site-specific mutagenesis without phenotypic selection
Methods Enzymol.
Antifreeze proteins of teleost fishes
Annu. Rev. Physiol.
Glycoproteins as biological antifreeze agents in antarctic fishes
Science
Fish antifreeze protein and the freezing and recrystallization of ice
Nature
Antifreeze proteins modify the freezing process in planta
Plant Physiol.
Heat-stable antifreeze protein from grass
Nature
A carrot leucine-rich-repeat protein that inhibits ice recrystallization
Science
Ice-binding proteins from sea ice diatoms (Bacillariophyceae)
J. Phycol.
Adsorption inhibition as a mechanism of freezing resistance in polar fishes
Proc. Natl Acad. Sci. USA
Structural biology. Adding to the antifreeze agenda
Nature
Dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations
J. Am. Chem. Soc.
Anchored clathrate waters bind antifreeze proteins to ice
Proc. Natl Acad. Sci. USA
Cited by (110)
Diffusion Attachment Model for Long Helical Antifreeze Proteins to Ice
2022, BiomacromoleculesCold acclimation and prospects for cold-resilient crops
2021, Plant StressAnti freeze proteins (Afp): Properties, sources and applications – A review
2021, International Journal of Biological MacromoleculesCitation Excerpt :An extremely heat stable protein LpAFP was isolated from a perennial ryegrass Lolium perenne showing low TH activity with high IRI activity. This AFP has 118 amino acid residues that fold into left handed β-coil structure [109,110]. Six AFPs with molecular mass varying from 15 to 38 kDa were isolated from the leaf of Secale cereale and they were found to be in homology with PR-proteins endoglucanase, endochitinase and thaumatin like proteins [111].
Preparation of biological antifreeze protein-modified emulsified asphalt coating and research on its anti-icing performance
2021, Construction and Building MaterialsCitation Excerpt :In this study, AFP was observed to have a good thermal hysteresis effect, recrystallisation inhibition effect, and ice crystal shaping effect, effectively inhibiting the growth of ice crystals. Owing to its non-polluting, renewable, and edible properties, AFP has been applied in many fields, including the food industry, agriculture, and industry, and has great application prospects [27–35]. Xu [36] described the structure, mechanism of action, and application prospects of AFP in aquatic animals.