THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 39, pp. 29030–29041, September 29, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Denmotoxin, a Three-finger Toxin from the Colubrid Snake Boiga dendrophila (Mangrove Catsnake) with Bird-specific Activity* Received for publication, June 19, 2006, and in revised form, July 21, 2006 Published, JBC Papers in Press, July 24, 2006, DOI 10.1074/jbc.M605850200 Joanna Pawlak‡, Stephen P. Mackessy§, Bryan G. Fry‡1, Madhav Bhatia¶, Gilles Mourierʈ, Carole Fruchart-Gaillardʈ, Denis Serventʈ, Rene´eMe´nezʈ, Enrico Sturaʈ, Andre´Me´nezʈ, and R. Manjunatha Kini‡**2 From the ‡Department of Biological Sciences, Faculty of Science, and the ¶Department of Pharmacology, Faculty of Medicine, National University of Singapore, Singapore 117543, the §School of Biological Sciences, University of Northern Colorado, Greeley, Colorado 80639-0017, the ʈDe´partement d’Inge´nierie et d’Etudes des Prote´ines, Commissariat a` l’Energie Atomique, Gif-sur-Yvette Cedex 91191, France, and the **Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Boiga dendrophila (mangrove catsnake) is a colubrid snake Three-finger toxins (3FTXs)3 form one of the most abun- that lives in Southeast Asian lowland rainforests and man- dant, well recognized families of snake venom proteins. They grove swamps and that preys primarily on birds. We have share a similar structure and are characterized by three Downloaded from isolated, purified, and sequenced a novel toxin from its ␤-stranded finger-like loops, emerging from a globular core venom, which we named denmotoxin. It is a monomeric and stabilized by four conserved disulfide bridges. An addi- polypeptide of 77 amino acid residues with five disulfide tional disulfide linkage may sometimes be present in the first bridges. In organ bath experiments, it displayed potent (non-conventional toxins) or second (long-chain ␣-neuro- postsynaptic neuromuscular activity and irreversibly inhib- toxins and ␬-toxins) loop (1–5). All 3FTXs are monomers ited indirectly stimulated twitches in chick biventer cervicis except for ␬-toxins, which are noncovalent homodimers iso- www.jbc.org nerve-muscle preparations. In contrast, it induced much lated from Bungarus venoms. Minor structural differences in smaller and readily reversible inhibition of electrically the three-finger fold, viz. the number of ␤-strands, overall induced twitches in mouse hemidiaphragm nerve-muscle morphology of the loops, and differential lengths of turns or ␣ ␤␥␦ by guest on October 9, 2006 preparations. More precisely, the chick muscle 1 -nico- C-terminal tails (6), lead to the recognition of varied targets tinic acetylcholine receptor was 100-fold more susceptible and modulate the toxicity and specificity (7). Hence, 3FTXs ␣ compared with the mouse receptor. These data indicate that affect a broad range of molecular targets, including 1-nico- denmotoxin has a bird-specific postsynaptic activity. We tinic acetylcholine receptors (nAChRs; short- and long- ␣ ␣ ␣ chemically synthesized denmotoxin, crystallized it, and chain -neurotoxins), 7-nAChRs (long-chain -neurotox- ˚ ␣ ␣ ␬ solved its crystal structure at 1.9 A by the molecular replace- ins), and 3- and 4-nAChRs ( -toxins) (4, 5); muscarinic ment method. The toxin structure adopts a non-conventional acetylcholine receptors (muscarinic toxins) (8); L-type cal- three-finger fold with an additional (fifth) disulfide bond in cium channels (calciseptine and FS2 toxin) (9, 10); integrin ␣ ␤ ␣ ␤ the first loop and seven additional residues at its N terminus, IIb 3 (dendroaspin) (11, 12); integrin v 3 (cardiotoxin A5) which is blocked by a pyroglutamic acid residue. This is the (13); acetylcholinesterase (fasciculins) (14); phospholipids first crystal structure of a three-finger toxin from colubrid and glycosphingolipids (cardiotoxins) (15); and blood coag- snake venom and the first fully characterized bird-specific ulation protein factor VIIa (16). As the interaction with such toxin. Denmotoxin illustrates the relationship between toxin a broad spectrum of target proteins results in a variety of specificity and the primary prey type that constitutes the pharmacological effects, the understanding of the physiolog- snake’s diet. ical role and mechanism of action supported by the architec- ture of 3FTXs could be of great value in the development of novel research tools or rational drug design. For instance, * This work was supported by a Biomedical Research Council grant from the Agency for Science, Technology and Research, Singapore. The costs studies with ␣-bungarotoxin led to a detailed characteriza- of publication of this article were defrayed in part by the payment of tion of muscle nAChR, helping to elucidate the fundamental page charges. This article must therefore be hereby marked “advertise- underlying mechanisms involved in neuromuscular trans- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. missions (17). The atomic coordinates and structure factors (code 2H5F) have been deposited in 3FTXs were thought to occur only in the venoms of the snake the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, family Elapidae. But recently, our laboratory reported the first Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) DQ366293. 1 Present address: Australian Venom Research Unit, School of Medicine, Uni- 3 The abbreviations used are: 3FTXs, three-finger toxins; nAChRs, nicotinic versity of Melbourne, Parkville, Victoria 3010, Australia. acetylcholine receptors; RP-HPLC, reversed-phase high pressure liquid 2 To whom correspondence should be addressed: Protein Science Lab., Dept. chromatography; RT, reverse transcription; Fmoc, N-(9-fluorenyl)methoxy- of Biological Sciences, Faculty of Science, National University of Singapore, carbonyl; CBCM, chick biventer cervicis muscle; MHD, mouse hemidia- Science Dr. 4, Singapore 117543. Tel.: 65-6516-5235; Fax: 65-6779-2486; phragm muscle; ESI/MS, electrospray ionization mass spectrometry; E-mail: [email protected]. AChBP, acetylcholine-binding protein. 29030 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 39•SEPTEMBER 29, 2006 Pharmacological and Structural Characterization of Denmotoxin colubrid snake venom toxin (␣-colubritoxin from Coelo- France). Piperidine, N-methylpyrrolidone, dichlorometh- gnathus radiatus), a reversible antagonist of chick muscle ane, methanol, trifluoroacetic acid, and tert-butyl methylox- nAChRs (18). The large polyphyletic family Colubridae consists ide were from SDS (Peypin, France). Tris(2-carboxyeth- of approximately two-thirds of the described species of the yl)phosphine hydrochloride was from Pierce. advanced snakes and includes Ͼ700 different venomous spe- Snake Venoms—B. dendrophila venom was extracted as cies (19, 20). Colubrid snakes possess a venom gland (the described previously (23) from snakes originating from Duvernoy’s gland) with a common duct extending to the pos- Sulawesi, Indonesia. Bungarus multicinctus venom was terior lingual region of the maxilla, commonly associated with obtained from Venom Supplies Pty. Ltd. (Tanunda, South grooved or enlarged posterior maxillary teeth. Relatively little is Australia, Australia), and ␣-bungarotoxin was purified using known of the chemistry, toxicology, and immunology of colu- a standard method (24). brid venoms, and a more thorough investigation of colubrid Animals—Swiss-Webster albino mice (32 Ϯ 2 g of body venom components holds great promise for discovering new weight) were purchased from the Laboratory Animals Cen- bioactive compounds (21, 22). ter of the National University of Singapore (Sembawang, In this work, we report the isolation, purification, and Singapore) and kept in the Department of Pharmacology pharmacological and structural characterization of a novel animal holding unit. Paper pellet bedding and food was pur- bird-specific neurotoxin, denmotoxin, one of the major chased from Glen Forrest Stockfeeders (Glen Forrest, West- venom components of the mangrove catsnake (Boiga den- ern Australia, Australia). Food and water were available ad drophila). Denmotoxin produced a virtually irreversible libitum. The animals were housed five per cage in a light- ␣ ␤␥␦ blockage of chick muscle 1 -nAChRs, whereas it pro- controlled room (12-h light/dark cycles) at 23 °C and 60% duced a much weaker, readily reversible blockage of mouse relative humidity. Downloaded from ␣ ␤␥␦ muscle 1 -nAChRs. In addition to pharmacological and 6–10-day-old domestic chicks (Gallus domesticus) were biochemical studies, we have established a chemical synthe- purchased from an agricultural farm in Singapore and delivered sis and refolding protocol for synthetic denmotoxin and on the day of experimentation. Experimental animals were solved the high resolution crystal structure of the protein. killed by exposure to 100% CO2. Denmotoxin has great potential to become a model for a Protein Purification—Previously, we had performed liquid www.jbc.org better understanding of the mechanisms of species specific- chromatography/mass spectrometry crude venom profiling of ity as well as reversible binding of venom neurotoxins. B. dendrophila venom (25) to identify venom complexity and to predict the composition of venom proteins. A major protein