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

Abstract

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.

A Unique Capsular Polysaccharide Structure from the Psychrophilic Marine Bacterium Colwellia psychrerythraea 34H That Mimics Antifreeze (Glyco)proteins

“…[55][56][57] For example, antifreeze glycoproteins (AFGPs) isolated from fish blood plasma consist of a regular tripeptide sequence of Ala-Ala-Thr with a disaccharide fragment (-D-galactosyl-(1-3)--D-galactosamine) linked to the threonine residue. [58][59][60][61] It is noteworthy that the polysaccharide described here displays a structural feature very close to that present in AFGPs. Actually, a -N-acetyl-galactosamine is substituted by an -galacturonosyl residue bearing an amide-linked threonine.…”

Flies expand the repertoire of protein structures that bind ice

“…The bacterial ice adhesion domain of MpAFP was correctly modeled as a β-solenoid based on the RTX repeats of an alkaline protease, which predicted that one of the two rows of internal Ca 2+ ions was absent and replaced by hydrophobic packing (8,30). Another AFP structure predicted de novo is that of the inquisitor beetle (RiAFP), which, along with inchworm AFP (iwAFP), was modeled as a flat, silklike β-solenoid (14). Although the iwAFP structure has not yet been experimentally determined, the recently solved structure of the similar RiAFP confirms the accuracy of the modeled structure (13).…”

“…Cleavage of this pro sequence from the mature AFP occurs at the C-terminal side of an arginine, which is a known cleavage site for trypsin-like proteases that are present in fly hemolymph (43). Pro sequences are present in the winter flounder type I AFP, sea raven type II AFP, and iwAFP but their functions are unknown (14,44,45). They may assist in proper protein folding and then become cleaved during or after export from the cell.…”

“…The crystal structures of four arthropod AFPs have been solved (10)(11)(12)(13), and two more structures have been modeled with confidence (14,15). The five arthropod AFPs are not homologous to each other.…”

Modeling repetitive, non‐globular proteins

“…Many of the IBPs with known structures have been determined experimentally to have sequences that are composed of multiple short repeats (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) residues in length) that in turn form repeating structural motifs. These structural motifs include a-helices and polyproline type II helices, but the majority of repetitive IBPs form a b-solenoid structure.…”