Ice-binding Motifs Research Articles

Brachypodium Antifreeze Protein Gene Products Inhibit Ice Recrystallisation, Attenuate Ice Nucleation, and Reduce Immune Response.

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, Brachypodium AFP genes encode two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models which indicate that ice-binding motifs on the ~13 kDa AFPs can “spoil” nucleating arrays on the ~120 kDa bacterial ice nucleating proteins used to form ice at high sub-zero temperatures. These models are consistent with the experimentally demonstrated decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Additionally, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22). Structural models suggested that this was due to the affinity of the LRR domains to flg22. Overall, it is remarkable that the Brachypodium genes play multiple distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins’ ability to adsorb to ice as well as to attenuate bacterial ice nucleation and the host immune response.

Inhibition of Ice Recrystallization by Nanotube-Forming Cyclic Peptides.

While most native ice-binding proteins are rigid, artificial (macro)molecular ice-binders are usually flexible. Realizing a regular array with precisely positioned ice-binding motifs on synthetic proteins, (macro)molecular ice-binders are thus challenging. Here, we exploit the predictable assembly of cyclic peptides into nanotubes as a starting point to prepare large, rigid ice-binders bearing an ice-binding site that is found in hyperactive ice-binding proteins in insects. First, we designed, synthesized, and purified cyclic octapeptide Lys2CP8 bearing a TaT motif to promote ice binding and investigated their solution assembly and activity using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, light scattering (LS), cryogenic transmission electron microscopy (cryo-TEM), and ice recrystallization inhibition (IRI) assays. The cyclic peptide Lys2CP8 was synthesized in good yield using Fmoc chemistry and purified by reversed-phase HPLC. Upon dissolution in aqueous solutions, Lys2CP8 was observed to assemble in a pH- and concentration-dependent manner into objects with nanoscopic dimensions. LS revealed the presence of small and large aggregates at pH 3 and 11, held together through a network of intermolecular antiparallel β-sheets as determined by FTIR and CD spectroscopy. Cryo-TEM revealed the presence of one-dimensional objects at pH 3 and 11. These are mostly well-dispersed at pH 3 but appear to bundle at pH 11. Interestingly, the pH-dependent self-assembly behavior translates into a marked pH dependence of IRI activity. Lys2CP8 is IRI-active at pH 3 while inactive at pH 11 hypothetically because the ice-binding sites are inaccessible at pH 11 due to bundling.

Ice-Nucleating and Antifreeze Proteins Recognize Ice through a Diversity of Anchored Clathrate and Ice-like Motifs.

Cold-adapted organisms produce antifreeze and ice-nucleating proteins to prevent and promote ice formation. The crystal structure of hyperactive bacterial antifreeze protein (AFP) MpAFP suggests that this protein binds ice through an anchored clathrate motif. It is not known whether other hyperactive AFPs and ice-nucleating proteins (INPs) use the same motif to recognize or nucleate ice. Here we use molecular simulations to elucidate the ice-binding motifs of hyperactive insect AFPs and a model INP of Pseudomonas syringae. We find that insect AFPs recognize ice through anchored clathrate motifs distinct from that of MpAFP. By performing simulations of ice nucleation by PsINP, we identify two distinct ice-binding sites on opposite sides of the β-helix. The ice-nucleating sequences identified in the simulations agree with those previously proposed for the closely related INP of Pseudomonas borealis based on the structure of the protein. The simulations indicate that these sites have comparable ice-nucleating efficiency, but distinct binding motifs, controlled by the amino acid sequence: one is an anchored clathrate and the other ice-like. We conclude that anchored clathrate and ice-like motifs can be equally effective for binding proteins to ice and promoting ice nucleation.

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm , of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 μmol.

Functional evaluation of candidate ice structuring proteins using cell-free expression systems

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.

Hyperactive antifreeze proteins from longhorn beetles: Some structural insights

This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of β-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these β-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall β-helical structure with the ice-binding motifs stacked in a β-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a β-helical conformation.

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.

Ice recrystallisation inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hairgrassDeschampsia antarcticaE. Desv.

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

Enhancing the activity of a beta-helical antifreeze protein by the engineered addition of coils.

The effectiveness of natural antifreeze proteins in inhibiting the growth of a seed ice crystal seems to vary with protein size. Here we have made use of the extreme regularity of the beta-helical antifreeze protein from the beetle Tenebrio molitor to explore systematically the relationship between antifreeze activity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils of the beta-helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, the seven-coil parent antifreeze protein, we have made a series of constructs with 6-11 coils. Misfolded forms of these antifreezes were removed by ice affinity purification to accurately compare the specific activity of each construct. There was a 10-100-fold gain in activity upon going from six to nine coils, depending on the concentration that was compared. Activity was maximal for the nine-coil construct, which gave a freezing point depression of 6.5 C degrees at 0.7 mg/mL, but actually decreased for the 10- and 11-coil constructs. This small loss in activity might result from the accumulation of a slight mismatch between the spacing of the ice-binding threonine residues and the O atoms of the ice lattice.

Biochemical Adaptation to the Freezing Environment - the Biology of Fish Antifreeze Proteins

Many organisms are known to survive in icy environments. These include both over wintering terrestrial insects and plants as well the marine fish inhabiting high latitudes. The adaptation of these organisms is both a fascinating and important topic in biology. Marine teleosts in particular, can encounter ice-laden seawater that is approximately <TEX>$1^{\circ}C$</TEX> colder than the colligative freezing point of their body fluids. These animals produce a unique group of proteins, the antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that absorb the ice nuclei and prevent ice crystal growth. Presently, there are at least four different AFP types and one AFGP type that are isolated from a wide variety of fish. Despite their functional similarity, there is no apparent common protein homology or ice-binding motifs among these proteins, except that the surface-surface complementarity between the protein and ice are important for binding. The remarkable diversity of these proteins and their odd phylogenetic distribution would suggest that these proteins might have evolved recently in response to sea level glaciations just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Winter flounder, Pleuronectes americanus, has been used as a popular model to study the regulation of AFP gene expression. It has a built-in annual cycle of AFP expression controlled negatively by the growth hormone. The signal transduction pathways, transcription factors and promoter elements involved in this process have been studied in our laboratory and these studies will be presented.

A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana.

The freeze-intolerant insect, Choristoneura fumiferana (spruce budworm), produces multiple antifreeze protein (AFP) isoforms for protection during the overwintering stage. We now report the cloning of AFP genes from insects; Afp-Lu1 encodes a approximately 9-kDa AFP isoform, and Afp-Iu1 encodes a approximately 12-kDa AFP isoform. Both CfAFP genes have similar structures with a single 3- to 3.6-kb intron interrupting the coding region. The second exon of an additional CfAFP gene, 2.7a, encoding a new approximately 9-kDa isoform, was found 3.7 kb upstream of Afp-Lu1 and demonstrates that some AFP family members are linked in tandem. This gene appears to encode an AFP with 68-76% identity to previously isolated CfAFPs. With its eight Cys residues necessary for disulfide bonding and five perfectly conserved 'Thr button' (Thr-Xaa-Thr) ice-binding motifs, it can be modeled as a functional AFP. Southern blot analysis shows that there are approximately 17 genes in this AFP family, with each of the isoforms represented by two to five gene copies. Transcript accumulation from Afp-Lu1 and Afp-Iu1 (or closely related genes) was maximal during the overwintering stage, while 2.7a transcripts were only detected in first instars, larvae that are normally found only in the summer. Contrary to expectations, this differential expression demonstrates that CfAFP gene family transcripts are primarily regulated during development, rather than by seasonally low temperatures.

Antifreeze proteins of teleost fishes.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.

Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.

Studies of a putative ice-binding motif in winter flounder skin-type anti-freeze polypeptide

Winter flounder contains two distinct anti-freeze protein isoforms, which are the liver-type extracellular anti-freeze proteins and the skin-type intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit lower anti-freeze activities than the liver-type isoforms and this might be due to their lacking complete ice-binding motifs. One of the skin-type anti-freeze proteins, skin-type anti-freeze protein-3, does contain putative overlapping ice-binding motifs with the sequences ‘-K—DT-’ and ‘-DT–K-’. Synthetic anti-freezes containing 0–3 repeats of the ‘-DT–K-’ motif were tested for stability and activity. Loss of the single ‘-DT–K-’ of skin-type anti-freeze protein-3 increases the anti-freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0°C.

Alternative Roles for Putative Ice-Binding Residues in Type I Antifreeze Protein

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.

Ice-binding structure and mechanism of an antifreeze protein from winter flounder

Antifreeze proteins provide fish with protection against the freezing effect of polar environments by binding to ice surfaces and inhibiting growth of ice crystals. We present the X-ray crystal structure at 1.5 A resolution of a lone alpha-helical antifreeze protein from winter flounder, which provides a detailed look at its ice-binding features. These consist of four repeated ice-binding motifs, the side chains of which are inherently rigid or restrained by pair-wise side-chain interactions to form a flat binding surface. Elaborate amino- and carboxy-terminal cap structures are also present, which explain the protein's rich alpha-helical content in solution. We propose an ice-binding model that accounts for the binding specificity of the antifreeze protein along the <0112> axes of the (2021) ice planes.