The Thr- and Ala-Rich Hyperactive Antifreeze Protein from Inchworm Folds as a Flat Silk-like β-Helix

Inchworm larvae of the pale beauty geometer moth, Campaea perlata, exhibit strong (6.4 °C) freezing point depression activity, indicating the presence of hyperactive antifreeze proteins (AFPs). We have purified two novel Thr- and Ala-rich AFPs from the larvae as small (∼3.5 kDa) and large (∼8.3 kDa) variants and have cloned the cDNA sequences encoding both. They have no homology to known sequences in current BLAST databases. However, these proteins and the newly characterized AFP from the Rhagium inquisitor beetle both contain stretches rich in alternating Thr and Ala residues. On the basis of these repeats, as well as the discontinuities between them, a detailed structural model is proposed for the 8.3 kDa variant. This 88-residue protein is organized into an extended parallel-stranded β-helix with seven strands connected by classic β-turns. The alternating β-strands form two β-sheets with a thin core composed of interdigitating Ala and Ser residues, similar to the thin hydrophobic core proposed for some silks. The putative ice-binding face of the protein has a 4 × 5 regular array of Thr residues and is remarkably flat. In this regard, it resembles the nonhomologous Thr-rich AFPs from other moths and some beetles, which contain two longer rows of Thr in contrast to the five shorter rows in the inchworm protein. Like that of some other hyperactive AFPs, the spacing between these ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice

Representative type I AFPs showing their diversity both within species and between species.

<p>Symbols and coloring are as in Fig. 2A. A) Alignment of smaller skin and circulatory isoforms from winter flounder liver (WF-Liv, M63478.1) and skin (WF-skin, M63478.1), cunner (JF937681.2), shorthorn sculpin (SHS) SS-8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Baardsnes2" target="_blank">[38]</a>and Liv5, longhorn sculpin (LHS, AF306348.1) and cunner (JF937681.2). Only WF-Liv possesses a signal peptide (lower case font, difference relative to WF-hyp in blue) and pro-peptide (italics) which is shown on the line above the mature AFP sequence. Amino acids encoded by codons interrupted by an intron in the cunner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Hobbs2" target="_blank">[71]</a> and flounder liver sequences are indicated with a wavy underline. The intron within the flounder skin gene lies within the 5′ UTR. B) Sequence of the hyperactive type I AFP from winter flounder (WF-hyp, EU188795.1) denoted as in Fig. 2A. This circulating isoform is dimeric and possesses a signal peptide (lower case font) but no pro-sequence. C) Sequence of the two atypical type I AFPs of intermediate length from shorthorn sculpin skin (SHS-skin, AF305502.1) and dusky snailfish (AY455863.1). Thr is seldom found in position <i>i</i> of the 11 aa <i>i</i>, <i>i</i>+4, <i>i</i>+8 pattern of ice-binding residues and this pattern is not necessarily continuous in these longer AFPs. Neither AFP possesses a signal peptide or prosequence.</p

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein–protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein–protein crystal contacts with few surface waters. Here we have changed the protein–protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters

Sculpin AFPs.

<p>A) Alignment of Sculpin AFPs. New sequences from shorthorn sculpin cDNAs from liver (Liv), larvae (Lar) or genomic DNA (G) are compared to known shorthorn skin (Skin) and longhorn skin (LHS) sequences. As the deduced peptide sequences are low complexity, they were aligned based on the DNA sequence alignment, which is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285.s001" target="_blank">Fig. S1</a> along with the accession numbers. Thr is highlighted light green and other polar residues are highlighted dark green with white font. Basic residues are highlighted cyan (Lys) or blue (Arg), acidic are highlighted red in black (Asp) or white (Glu) font, hydrophobic residues (except Ala) are highlighted gray and exceptional residues (Pro and Gly) are highlighted yellow. B) Dot matrix comparisons of selected sculpin isoforms. A line indicates a match of at least 9 out of 10 bases. Coding regions are denoted by blue bars.</p

Properties of the type I AFPs from the four groups of fish.

a<p>The sequence of a larger circulating isoform is unknown but an accurate mass and rough composition were determined from an enriched sample <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Evans4" target="_blank">[66]</a>.</p>b<p>Longhorn sculpin skin and circulating isoforms are highly similar, but one shorthorn sculpin cDNA from skin encodes a distinct isoform.</p

Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions

<div><p>Alanine-rich α-helical (type I) antifreeze proteins (AFPs) are produced by a variety of fish species from three different orders to protect against freezing in icy seawater. Interspersed amongst and within these orders are fishes making AFPs that are completely different in both sequence and structure. The origin of this variety of types I, II, III and antifreeze glycoproteins (AFGPs) has been attributed to adaptation following sea-level glaciations that occurred after the divergence of most of the extant families of fish. The presence of similar types of AFPs in distantly related fishes has been ascribed to lateral gene transfer in the case of the structurally complex globular type II lectin-like AFPs and to convergent evolution for the AFGPs, which consist of a well-conserved tripeptide repeat. In this paper, we examine the genesis of the type I AFPs, which are intermediate in complexity. These predominantly α-helical peptides share many features, such as putative capping structures, Ala-richness and amphipathic character. We have added to the type I repertoire by cloning additional sequences from sculpin and have found that the similarities between the type I AFPs of the four distinct groups of fishes are not borne out at the nucleotide level. Both the non-coding sequences and the codon usage patterns are strikingly different. We propose that these AFPs arose via convergence from different progenitor helices with a weak affinity for ice and that their similarity is dictated by the propensity of specific amino acids to form helices and to align water on one side of the helix into an ice-like pattern.</p></div

Amino acids encoded in alternative reading frames by adjacent Ala codons.

<p>Amino acids encoded in alternative reading frames by adjacent Ala codons.</p

Evolutionary relationships between AFP-producing fishes and the similarities between type I AFPs.

<p>A) Phylogenetic relationships (not to scale) among AFP-producing fish from analysis of complete mitochondrial genomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Miya1" target="_blank">[67]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Lavoue1" target="_blank">[69]</a> or selected nuclear and mitochondrial sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Matschiner1" target="_blank">[45]</a>. Estimated divergence times (Ma, some with 95% highest posterior density limits) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Matschiner1" target="_blank">[45]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Steinke1" target="_blank">[47]</a> are shown at some nodes. Species names are colored by AF(G)P type as indicated on the right. Representative ribbon structures are shown for types II, III, and I AFPs (PDB 2PY2, 1HG7, 1WFA from top to bottom, red = helix, green = strand, gray = coil). The colored bars at the bottom indicate climate differences marked by the presence (blue) or absence (red) of large ice sheets. Common names of representative AFP-producing fish are indicated but their scientific names are as follows; herring (<i>Clupea harengus</i>), Arctic cod (<i>Boreogadus saida</i>), cunner (<i>Tautogolabrus adspersus</i>), ocean pout (<i>Zoarces americanus</i>), Atlantic snailfish (<i>Liparis atlanticus</i>), dusky snailfish (<i>Liparis gibbus</i>), sea raven (<i>Hemitripterus americanus</i>), longhorn sculpin (<i>Myoxocephalus octodecemspinosus</i>), shorthorn sculpin (<i>Myoxocephalus scorpius</i>), Antarctic toothfish (<i>Dissostichus mawsoni</i>), winter flounder (<i>Pseudopleuronectes americanus</i>) and rainbow smelt (<i>Osmerus mordax</i>). B) Alignment of representative type I skin AFPs from three fishes from three separate orders (winter flounder (M63478.1), longhorn sculpin (AF306348.1) and cunner (JF937681.2). Potential or known ice-binding residues within the 11-aa repeat that show an <i>i</i>, <i>i</i>+4, <i>i</i>+8 spacing pattern are indicated with plus symbols (Ala) and number symbols (Thr) with asterisks denoting residues that are identical in all sequences. Acidic and basic residues are in red and blue font respectively, with Ala highlighted yellow and Thr in white font with black highlighting. Potential helix-stabilizing salt bridges consisting of basic and acidic residues with the more effective <i>i</i>, <i>i</i>+4 separation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Marqusee1" target="_blank">[70]</a> are double underlined. The cunner isoform is also found in blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Evans2" target="_blank">[42]</a>.</p

Ala codon usage in type I AFPs.

<p>The height of the color bars represents the fraction of each of the Ala codons in each dataset or sequence. The cDNAs used encoded the following AFPs; WF–hyp, LHS, SHS–skin, Atlantic snailfish AFP and cunner AFP. The number of non-AFP sequences used for each group is as follows: winter flounder, 70; longhorn sculpin, 10; cunner, 10 and four species in the snailfish family (Liparidae), 8. The accession numbers for these sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285.s005" target="_blank">Text S1</a>.</p

Dot matrix comparisons of type I cDNAs from the four different groups of fishes.

<p>A line indicates a match of at least 9 out of 12 bases with red indicating a sense/antisense match. The blue bars denote the coding region (signal peptides excluded). These sequences correspond to those shown in Fig. 3.</p