Hyperactive antifreeze proteins from longhorn beetles: Some structural insights Erlend Kristiansen, Casper Wilkens 1 , Bjarne Vincents 2 , Dennis Friis, Anders Blomkild Lorentzen, Håvard Jenssen, Anders Løbner-Olesen, Hans Ramløv ⇑ Institute of Science, Systems and Models, Roskilde University Center, DK-4000 Roskilde, Denmark article info Article history: Received 12 July 2012 Received in revised form 7 September 2012 Accepted 10 September 2012 Available online 21 September 2012 Keyword: Antifreeze protein Rhagium Structure Cloning Expression Beetle abstract 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 character- ized 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 b- sheet and low levels of a-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these b-sheets within the putative ice-binding motifs and the central parts of the seg- ments separating them, consistent with an overall b-helical structure with the ice-binding motifs stacked in a b-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a b-helical conformation. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Many organisms spend extended periods, in some cases their entire lives, in environments so cold that their body temperatures fall below the equilibrium freezing temperature of their body flu- ids. A number of these species have evolved so-called antifreeze proteins (AFPs) or glycoproteins (AFGPs) that they accumulate in their body fluids. Such AF(G)Ps are characterized by their ability to prevent ice from expanding when a solution containing ice crystals is cooled below its melting temperature. Fish that occupy ice-laden polar waters, many species of insects, collembolans and spiders are known to produce molecules that prevent the growth of ice crystals (Raymond and deVries, 1977; Zachariassen and Hus- by, 1982; Duman et al., 2004; Pentelute et al., 2008). The ability of AF(G)Ps to prevent ice growth and their frequent occurrence in cold hardy organisms of such diverse origin suggests that they are an effective protective measure against internal ice formation. Consistent with this notion of function, AFPs have been shown both to neutralize the ability of structures to trigger freezing (Olsen and Duman, 1997, 2002) and to prevent external ice from penetrat- ing through the body surface (Gehrken, 1992; Olsen et al., 1998). Other organisms including plants and even some yeast and bacte- ria that inhabit cold regions also produce proteins that may inter- act with ice (Duman and Olsen, 1993; Gilbert et al., 2005; Lee et al., 2012) although their physiological role remains unclear. The reported antifreeze activity varies from about 1 °C in polar fish plasma to commonly 4–7 °C in the hemolymph of terrestrial insects and spiders. Insect AFPs are reportedly 10–100 times more potent antifreeze agents than AF(G)Ps from polar fishes at equimo- lar concentrations, a characteristic ascribed to the harsher thermal environment on land. The term ‘‘hyperactive’’ is often used to de- scribe the strongly augmented potency of insect AFPs relative to most other AF(G)Ps. According to the conventional view, all AF(G)Ps are thought to prevent ice growth as the result of becom- ing irreversibly adsorbed onto the surface of the ice crystal (Ray- mond and deVries, 1977). This concept is strongly supported by the structural match between the arrangement of water molecules in ice and parts of the surface of AFPs, coined their ice binding sites. Although a large number of insects are known to produce AFPs (Duman et al., 2004), surprisingly few have been isolated and char- acterized. Several species of the Lepidopteran genus Choristoneura express AFPs (Tyshenko et al., 2005). In these, a regular appearance of the amino acid triplet TxT is separated by 15 residue segments without any apparent repeated pattern. The ‘x’ position within the triplet is mostly occupied by Thr, Ser, Cys or Ala. The structure is stabilized by multiple disulphide bonds irregularly spaced apart (Graether et al., 2000; Tyshenko et al., 2005). Recently, Lin et al. (2011) reported that the primary structure of another lepidopter- an, the inch worm Campaea perlata, consists of the repeat sequence 0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2012.09.004 ⇑ Corresponding author. Address: Department of Science, Systems and Models, Roskilde University, Build. 18.1, Universitetsvej 1, P.O. Box 260, DK-4000 Roskilde, Denmark. Tel.: +45 46742739; fax: +45 46743011. E-mail address: hr@ruc.dk (H. Ramløv). 1 Present address: Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark. 2 Present address: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark. Journal of Insect Physiology 58 (2012) 1502–1510 Contents lists available at SciVerse ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys