Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.     

Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast

Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.     

Crystal structure of beta-helical antifreeze protein points to a general ice binding model

Reported here is the 2.3 A resolution crystal structure of spruce budworm (Choristoneura fumiferana) antifreeze protein (CfAFP), solved by single anomalous scattering. The structure reveals an extremely regular left-handed beta-helical platform consisting of 15-amino acid loops with a repetitive Thr-X-Thr motif displayed on one of the helix's three faces. This motif results in a two-dimensional array of threonine residues in an identical orientation to those in the nonhomologous, right-handed beta-helical beetle AFP from Tenebrio molitor (TmAFP). The CfAFP structure led us to reevaluate our ice binding model, and the analysis of three possible modes of docking gives rise to a binding mechanism based on surface complementarity. This general mechanism is applicable to both fish and insect AFPs.     

Evolution of Hyperactive,Repetitive Antifreeze Proteins in Beetles

Some organisms that experience subzero temperatures, such as insects, fish, bacteria, and plants, synthesize antifreeze proteins (AFPs) that adsorb to surfaces of nascent ice crystals and inhibit their growth. Although some AFPs are globular and nonrepetitive, the majority are repetitive in both sequence and structure. In addition, they are frequently encoded by tandemly arrayed, multigene families. AFP isoforms from the mealworm beetle, Tenebrio molitor, are extremely potent and inhibit ice growth at temperatures below −5°C. They contain a 12-amino acid repeat with the sequence TCTxSxxCxxAx, each of which makes up one coil of the β-helix structure. TxT motifs are arrayed to form the ice-binding surface in all three known insect AFPs: the homologous AFPs from the two beetles, T. molitor and Dendroides canadensis, and the nonhomologous AFP from the spruce budworm, Choristoneura fumiferana. In this study, we have obtained the cDNA and genomic sequences of additional T. molitor isoforms. They show variation in the number of repeats (from 6 to 10) which can largely be explained by recombination at various TCT motifs. In addition, phylogenetic comparison of the AFPs from the two beetles suggests that gene loss and amplification may have occurred after the divergence of these species. In contrast to a previous study suggesting that T. molitor genes have undergone positive Darwinian selection (selection for heterogeneity), we propose that the higher than expected ratio of nonsynonymous-to-synonymous substitutions might result from selection for higher AT content in the third codon position. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. John Oakeshott]     

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.     

Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity

Antifreeze proteins (AFPs) protect certain organisms from freezing by adhering to ice crystals, thereby preventing their growth. All AFPs depress the nonequilibrium freezing temperature below the melting point; however AFPs from overwintering insects, such as the spruce budworm (sbw) are 10-100 times more effective than most fish AFPs. It has been proposed that the exceptional activity of these AFPs depends on their ability to prevent ice growth at the basal plane. To test the hypothesis that the hyperactivity of sbwAFP results from direct affinity to the basal plane, we fluorescently tagged sbwAFP and visualized it on the surface of ice crystals using fluorescence microscopy. SbwAFP accumulated at the six prism plane corners and the two basal planes of hexagonal ice crystals. In contrast, fluorescently tagged fish type III AFP did not adhere to the basal planes of a single-crystal ice hemisphere. When ice crystals were grown in the presence of a mixture of type III AFP and sbwAFP, a hybrid crystal shape was produced with sbwAFP bound to the basal planes of truncated bipyramidal crystals. These observations are consistent with the blockage of c-axial growth of ice as a result of direct interaction of sbwAFP with the basal planes.     

Why does insect antifreeze protein from Tenebrio molitor produce pyramidal ice crystallites?

The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the orientations and molecular compositions of the primary ice surfaces that undergo growth rate reduction by the insect AFPs are obtained from first principles. Besides the basal face and primary prism, the ice surfaces engaged by insect AFPs include the specific ice pyramids produced by the insect AFP Tenebrio molitor (TmAFP). TmAFP-induced pyramids differ fundamentally from the ice pyramids produced by fish AFPs and antifreeze protein glycoproteins (AFPGs) as regards the ice surface configurations and the mode of interaction with the protein IBS. The molecular compositions of the TmAFP-induced pyramids are strongly bonded in two dimensions and have the constant face indices (101). In contrast, the molecular composition of the ice pyramids produced by fish AFPs and AFPGs are strongly bonded in only one direction and have variable face indices (h 0 l), none of which equal (101). The thus far puzzling behavior of the TmAFP in producing pyramidal crystallites is fully explained in agreement with experiment.     

Cold hardiness in relation to trace metal stress in the freeze-avoiding beetle Tenebrio molitor

The antifreeze proteins (AFPs) are a family of proteins characterised by their ability to inhibit the growth of ice. These proteins have evolved as a protection against lethal freezing in freeze avoiding species. Metal stress has been shown to reduce the cold hardening in invertebrates, but no study has investigated how this type of stress affects the production of AFPs. This study demonstrates that exposure to cadmium (Cd), copper (Cu) and zinc (Zn) reduces the normal developmental increase in AFP levels in Tenebrio molitor larvae reared under summer conditions. Exposure to winter conditions, however stimulated the production of AFPs in the metal exposed larvae, and raised the concentrations of AFPs to normal winter levels. The reduced level of AFPs in metal-stressed animals acclimated to summer conditions seems to arise from alterations in the normal gene expression of AFPs. The results indicate that metal exposure may cause freeze avoiding insects to become more susceptible to lethal freezing, as they enter the winter with lowered levels of AFPs. Such an effect cannot be revealed by ordinary toxicological tests, but may nevertheless be of considerable ecological importance.     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.     

Structure and dynamics of a beta-helical antifreeze protein

Antifreeze proteins (AFPs) protect many types of organisms from damage caused by freezing. They do this by binding to the ice surface, which causes inhibition of ice crystal growth. However, the molecular mechanism of ice binding leading to growth inhibition is not well understood. In this paper, we present the solution structure and backbone NMR relaxation data of the antifreeze protein from the yellow mealworm beetle Tenebrio molitor (TmAFP) to study the dynamics in the context of structure. The full (15)N relaxation analysis was completed at two magnetic field strengths, 500 and 600 MHz, as well as at two temperatures, 30 and 5 degrees C, to measure the dynamic changes that occur in the protein backbone at different temperatures. TmAFP is a small, highly disulfide-bonded, right-handed parallel beta-helix consisting of seven tandemly repeated 12-amino acid loops. The backbone relaxation data displays a periodic pattern, which reflects both the 12-amino acid structural repeat and the highly anisotropic nature of the protein. Analysis of the (15)N relaxation parameters shows that TmAFP is a well-defined, rigid structure, and the extracted parameters show that there is similar restricted internal mobility throughout the protein backbone at both temperatures studied. We conclude that the hydrophobic, rigid binding site may reduce the entropic penalty for the binding of the protein to ice. The beta-helical fold of the protein provides this rigidity, as it does not appear to be a consequence of cooling toward a physiologically relevant temperature.     

Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.     

Solution Structure of an Antifreeze Protein CfAFP-501 from <Emphasis Type="Italic">Choristoneura fumiferana</Emphasis>

Antifreeze proteins (AFPs) are widely employed by various organisms as part of their overwintering survival strategy. AFPs have the unique ability to suppress the freezing point of aqueous solution and inhibit ice recrystallization through binding to the ice seed crystals and restricting their growth. The solution structure of CfAFP-501 from spruce budworm has been determined by NMR spectroscopy. Our result demonstrates that CfAFP-501 retains its rigid and highly regular structure in solution. Overall, the solution structure is similar to the crystal structure except the N- and C-terminal regions. NMR spin-relaxation experiments further indicate the overall rigidity of the protein and identify a collection of residues with greater flexibilities. Furthermore, Pro91 shows a cis conformation in solution instead of the trans conformation determined in the crystal structure.Structural data have been deposited at PDB (1Z2F) and Chemical shift data at BMRB (bmrb 6111).     

Computational study on the function of water within a beta-helix antifreeze protein dimer and in the process of ice-protein binding

Antifreeze proteins (AFPs) help many organisms protect themselves from freezing in subzero temperatures. The most active AFPs found to date are those from insects, which possess exceptionally regular beta-helical structures. On the ice-binding surface of these proteins, regularly arrayed water molecules are observed within the repeating Thr-Xxx-Thr motif, but the exact role of these water molecules remains unknown. In this work, we have employed a number of computational methods to examine the role of these water molecules in an AFP from Tenebrio molitor (TmAFP). Our investigation involved a combination of molecular and quantum mechanical approaches. Properties such as stability, interaction energy, orbital overlap, and conformational analysis of various systems, including TmAFP-water, TmAFP-water-ice, and TmAFP-ice, were systematically evaluated and compared. The regularly arrayed water molecules were found to remain associated with TmAFP before ice binding, demonstrating that they are an intrinsic part of the protein. These water molecules may assist TmAFP in the process of ice recognition and binding. However, after facilitating the initial stages of ice recognition and binding, these water molecules are excluded in the final formation of the AFP-ice complex. The departure of these water molecules enables a better two-dimensional match between TmAFP and ice. These results agree with experimental observations showing that although these water molecules are aligned with the ice-binding hydroxyl groups of Thr residues in one dimension, they are in fact positioned slightly off in the second dimension, making a good two-dimensional match impossible.     

Effect of a mutation on the structure and dynamics of an alpha-helical antifreeze protein in water and ice

One strategy of psychrophilic organisms to survive subzero temperature is to produce antifreeze protein (AFPs), which inhibit the growth of macromolecular ice. To better understand the binding mechanism, the structure and dynamics of several AFPs have been studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The results have shown that different organisms can use diverse structures (alpha-helix, beta-helix, or different globular folds) to achieve the same function. A number of studies have focused on understanding the relationship between the alpha-helical structure of fish type I AFP and its function as an inhibitor of ice growth. The results have not explained whether the 90% activity loss caused by the conservative mutation of two threonines to serines (Thr13Ser/Thr24Ser) is attributable to a change in protein structure in solution or in ice. We examine here the structure and dynamics of the winter flounder type I AFP and the mutant Thr13Ser/Thr24Ser in both solution and solid states using a wide range of NMR approaches. Both proteins remain fully alpha-helical at all temperatures and in ice, demonstrating that the activity change must therefore not be attributable to changes in the protein fold or dynamics but differences in surface properties.     

Predicting the beta-helix fold from protein sequence data.

A method is presented that uses beta-strand interactions to predict the parallel right-handed beta-helix super-secondary structural motif in protein sequences. A program called BetaWrap implements this method and is shown to score known beta-helices above non-beta-helices in the Protein Data Bank in cross-validation. It is demonstrated that BetaWrap learns each of the seven known SCOP beta-helix families, when trained primarily on beta-structures that are not beta-helices, together with structural features of known beta-helices from outside the family. BetaWrap also predicts many bacterial proteins of unknown structure to be beta-helices; in particular, these proteins serve as virulence factors, adhesins, and toxins in bacterial pathogenesis and include cell surface proteins from Chlamydia and the intestinal bacterium Helicobacter pylori. The computational method used here may generalize to other beta-structures for which strand topology and profiles of residue accessibility are well conserved.     

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3₁₀-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.     

A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold

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     

Antifreeze proteins in higher plants

Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. Recently, several AFPs have been isolated and characterized and five full-length AFP cDNAs have been cloned and characterized in higher plants. The derived amino acid sequences have shown low homology for identical residues. Theoretical and experimental models for structure of Lolium perenne AFP have been proposed. In addition, it was found that the hormone ethylene is involved in regulating antifreeze activity in response to cold. In this review, it is seen that the physiological and biochemical roles of AFPs may be important to protect the plant tissues from mechanical stress caused by ice formation.     

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.     

Structure and evolutionary origin of Ca(2+)-dependent herring type II antifreeze protein

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.