The Refined Crystal Structure of an Eel Pout Type III Antifreeze Protein RD1 at 0.62-Å Resolution Reveals Structural Microheterogeneity of Protein and Solvation

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 Å. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the “pretzel fold” includes four short β-strands and a 3₁₀ helix. There is a novel internal cavity of 45 ų surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.     

Two domains of RD3 antifreeze protein diffuse independently

Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.     

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.     

Characterization of alpha-fetoprotein in fetal striped dolphin (Stenella coeruleoalba): purification of protein product and molecular cloning of the corresponding transcript

Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring pregnancy in many mammalian species. However, characterization of AFP has not yet been undertaken in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical precipitation followed by a combination of immunoadsorbent column chromatography and gel filtration. The molecular masses of native and denatured dolphin AFP were estimated to be ～78,000 Da by gel filtration and ～68,000 Da by SDS-PAGE, respectively, representing typical masses reported for mammalian AFPs. In fetal serum, only the AFP band (～68,000 Da) appeared to be immunoreactive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the development of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more than 80% identity to those of other mammalian AFPs. To our knowledge, the present report represents the first identification and characterization of AFP from any cetacean species.     

In silico structural and functional modelling of Antifreeze protein (AFP) sequences of Ocean pout (Zoarces americanus, Bloch & Schneider 1801)

Antifreeze proteins (AFPs) are known to polypeptide components formed by certain plants, animals, fungi and bacteria which support to survive in sub-zero temperature. Current study highlighted the seven different antifreeze proteins of fish Ocean pout (Zoarces americanus), in which protein (amino acids sequence) were collected from National Centre for Biotechnology Information and finely characterized using several in silico tools. Such biocomputational techniques applied to figure out the physicochemical, functional and conformational characteristics of targeted AFPs. Multiple physicochemical properties such as Isoelectric Point, Extinction Coefficient and Instability Index, Aliphatic Index, Grand Average Hydropathy were calculated and analysed by ExPASy-ProtParam prediction web server. EMBOSS: pepwheel online tool was used to represent the protein sequences in a helical form. The primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The secondary structure of these proteins was calculated using SOPMA tool. SOSUI server and CYS_REC program also run for ideal prediction of transmembrane helices and disulfide bridges of experimental proteins respectively. The modelling of 3D structures of seven desired AFPs were executed by the homology modelling programmes; SWISS MODEL and ProSA web server. UCSF Chimera, Antheprot 3D, PyMOL and RAMPAGE were used to visualize and analysis of the structural variation of the predicted protein model. MEGA7.0.9 software used to know the phylogenetic relationship among these AFPs. These models offered excellent and reliable baseline information for functional characterization of the experimentally derived protein domain composition by using the advanced tools and techniques of Computational Biology.     

Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3.

RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees. This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface.     

The amino-acid sequence of a lectin from conger eel, Conger myriaster, skin mucus.

The amino-acid sequence of a beta-galactoside-binding lectin isolated from the skin mucus of the conger eel Conger myriaster was determined. The lectin (30 kDa) was composed of two identical subunits of 135 amino acid residues with N-acetylserine at the N-terminus and no half-cystinyl residue. It was a 30-34% sequence identical to vertebrate beta-galactoside-binding lectin and proved to be a member of the S-type lectin family.     

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.     

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.     

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.     

NMR analysis of type III antifreeze protein intramolecular dimer. Structural basis for enhanced activity

The structure of a new antifreeze protein (AFP) variant, RD3, from antarctic eel pout (Rhigophila dearborni) with enhanced activity has been determined for the first time by nuclear magnetic resonance spectroscopy. RD3 comprises a unique translational topology of two homologous type III AFP globular domains, each containing one flat, ice binding plane. The ice binding plane of the N domain is located approximately 3.5 A "behind" that of the C domain. The two ice binding planes are located laterally with an angle of 32 +/- 12 degrees between the planes. These results suggest that the C domain plane of RD3 binds first to the ice [1010] prism plane in the <0001> direction, which induces successive ice binding of the N domain in the <0101> direction. This manner of ice binding caused by the unique structural topology of RD3 is thought to be crucial for the significant enhancement of antifreeze activity, especially at low AFP concentrations.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Antipeptide antibodies to the porcine lactate dehydrogenase isoenzyme M4 active center fragment as a probe for the structural analysis of active centers of human lactate dehydrogenase isoforms

Antipeptide antibodies (AB) to the fragment of the active center of porcine lactate dehydrogenase M4 isoform were used for the analysis of antigenic properties and structural comparison of active centers of human lactate dehydrogenase isoforms. Selective precipitation of the M-subunit-containing isoforms using an immunoadsorbent based on antipeptide AB as well as selective inhibition of the enzymic activity of the M4 isoform by antipeptide AB testify to the specific binding of isoforms to antipeptide AB. The experimental results confirm the literary data on conformational changes in the structure of the active centers of corresponding human lactate dehydrogenase isoforms. The specific interaction of antipeptide AB with human lactate dehydrogenase isoforms suggests that the site of the amino acid sequence (residues 180-214) in both human and porcine M4 isoenzymes is immunochemically identical. The data obtained suggest that antipeptide AB are convenient probes for detecting differences (including minor ones) in the primary and spatial structure of enzymes.     

Azuki bean (Vigna angularis) protease inhibitors: isolation and amino acid sequences

Double-headed protease inhibitors I, IIa, and IIc (AB I, AB IIa, and AB IIc) have been purified from azuki beans "Takara" (Vigna angularis) by conventional chromatographic methods and their amino acid sequences have been determined. AB I, AB IIa, and AB IIc had molecular weights of 9,166, 8,661, and 8,756 daltons, consisting of 82, 78, 79 amino acid residues, respectively. The molecular weights of these inhibitors, determined by gel filtration at pH 8.0, were 18,000 for AB I and 17,000 for both AB IIa and AB IIc, indicating that the inhibitors are dimers. The inhibitors had isoelectric points of 4.7 (AB I), 6.8 (AB IIa), and 6.2 (AB IIc). AB I stoichiometrically inhibited both trypsin and chymotrypsin at a molar ratio of 1 : 1. On the other hand, AB IIa and AB IIc both inhibited trypsin at a molar ratio of about 1 : 2 and also inhibited chymotrypsin, though only weakly. Sequence comparison with other double-headed inhibitors indicated the reactive sites of AB IIa and AB IIc for trypsin to be Lys26-Ser27 and Arg53-Ser54, and those of AB I for trypsin and chymotrypsin to be Lys26-Ser27 and Tyr53-Ser54, respectively. The differences between AB IIa and AB IIc were that AB IIa lacked the C-terminal aspartic acid residue, and that Glu10 and Arg60 in AB IIa were replaced by Gln10 and His60 in AB IIc. A comparison between AB IIa and AB I revealed 25 variant amino acids among the 78 residues of AB IIa; further, Ab IIa lacked 4 amino acid residues in the C-terminal region of AB I.     

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.     

The occurrence of two types of collagen proalpha-chain in the abalone Haliotis discus muscle.

Acid-soluble collagens were prepared from connective tissues in the abalone Haliotis discus foot and adductor muscles with limited proteolysis using pepsin. Collagen preparation solubilized with 1% pepsin contained two types of alpha-chains which were different in their N-terminal amino acid sequences. Accordingly, two types of full-length cDNAs coding for collagen proalpha-chains were isolated from the foot muscle of the same animal and these proteins were named Hdcols (Haliotis discus collagens) 1alpha and 2alpha. The two N-terminal amino acid sequences of the abalone pepsin-solubilized collagen preparation corresponded to either of the two sequences deduced from the cDNA clones. In addition, several tryptic peptides prepared from the pepsin-solubilized collagen and fractionated by HPLC showed N-terminal amino acid sequences identical to those deduced from the two cDNA clones. Hdcols 1alpha and 2alpha consisted of 1378 and 1439 amino acids, respectively, showing the primary structure typical to those of fibril-forming collagens. The N-terminal propeptides of the two collagen proalpha-chains contained cysteine-rich globular domains. It is of note that Hdcol 1alpha completely lacked a short Gly-X-Y triplet repeat sequence in its propeptide. An unusual structure such as this has never before been reported for any fibril-forming collagen. The main triple-helical domains for both chains consisted of 1014 amino acids, where a supposed glycine residue in the triplet at the 598th position from the N-terminus was replaced by alanine in Hdcol 1alpha and by serine in Hdcol 2alpha. Both proalpha-chains of abalone collagens contained six cysteine residues in the carboxyl-terminal propeptide, lacking two cysteine residues usually found in vertebrate collagens. Northern blot analysis demonstrated that the mRNA levels of Hdcols 1alpha and 2alpha in various tissues including muscles were similar to each other.     

The primary structure of ribosomal protein L2 from Bacillus stearothermophilus

The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o-iodosobenzoic acid. The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B. stearothermophilus are identical to those of the protein from E. coli and 45% are identical to those found in the two chloroplast proteins. There are extended regions of totally conserved sequence at positions 54-58 (GGGHK), 81-86 (EYDPNR), and 224-230 (MNPVDHP) in all four proteins.     

Quantitative and qualitative analysis of type III antifreeze protein structure and function

Some cold water marine fishes avoid cellular damage because of freezing by expressing antifreeze proteins (AFPs) that bind to ice and inhibit its growth; one such protein is the globular type III AFP from eel pout. Despite several studies, the mechanism of ice binding remains unclear because of the difficulty in modeling the AFP-ice interaction. To further explore the mechanism, we have determined the x-ray crystallographic structure of 10 type III AFP mutants and combined that information with 7 previously determined structures to mainly analyze specific AFP-ice interactions such as hydrogen bonds. Quantitative assessment of binding was performed using a neural network with properties of the structure as input and predicted antifreeze activity as output. Using the cross-validation method, a correlation coefficient of 0.60 was obtained between measured and predicted activity, indicating successful learning and good predictive power. A large loss in the predictive power of the neural network occurred after properties related to the hydrophobic surface were left out, suggesting that van der Waal's interactions make a significant contribution to ice binding. By combining the analysis of the neural network with antifreeze activity and x-ray crystallographic structures of the mutants, we extend the existing ice-binding model to a two-step process: 1) probing of the surface for the correct ice-binding plane by hydrogen-bonding side chains and 2) attractive van der Waal's interactions between the other residues of the ice-binding surface and the ice, which increases the strength of the protein-ice interaction.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.