NMR Solution Structure of Butantoxin 1 S. Kent Holaday, Jr.,* Brian M. Martin,† Paul L. Fletcher, Jr.,‡ and N. Rama Krishna* ,2 *Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-2041; †Clinical Neuroscience Branch, NIMH, National Institutes of Health, Bethesda, Maryland 20892-4405; and ‡Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858 Received December 22, 1999, and in revised form April 7, 2000 The NMR structure of a new toxin, butantoxin (BuTX), which is present in the venoms of the three Brazilian scorpions Tityus serrulatus, Tityus bahien- sis, and Tityus stigmurus, has been investigated. This toxin was shown to reversibly block the Shaker B po- tassium channels (K d  660 nM) and inhibit the prolif- eration of T-cells and the interleukin-2 production of antigen-stimulated T-helper cells. BuTX is a 40 amino acid basic protein stabilized by the four disulfide bridges: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36, and Cys20-Cys38. The latter three are conserved among all members of the short-chain scorpion toxin family, while the first is unique to BuTX. The three-dimen- sional structure of BuTX was determined using 1 H- NMR spectroscopy. NOESY, phase sensitive COSY (PH-COSY), and amide hydrogen exchange data were used to generate constraints for molecular modeling calculations. Distance geometry and simulated an- nealing calculations were performed to generate a family of 49 structures free of constraint violations. The secondary structure of BuTX consists of a short 2 1 2 turn -helix (Glu15-Phe23) and a -sheet. The -sheet is composed of two well-defined antiparallel strands (Gly29-Met32 and Lys35-Cys38) connected by a type-I -turn (Asn33-Asn34). Residues Cys5-Ala9 form a qua- si-third strand of the -sheet. The N-terminal C2-C5 disulfide bridge unique to this toxin does not appear to confer stability to the protein. © 2000 Academic Press Key Words: butantoxin; 2D-NMR; solution structure; potassium channels. Scorpion venom is a complex mixture of small pro- teins, histamine, serotonin, enzymes, enzyme inhibi- tors, mucus, and other poorly characterized com- pounds. These small proteins interact specifically with the Na + ,K + , and Cl - channels of mammals and in- sects, and are responsible for the neurotoxic activity of scorpion venom (1). The Na + channel toxins are re- sponsible for the majority of toxicity associated with envenomation in humans (2). The K + channel toxins are less important medically. However, they are valu- able as tools for studying K + channel function and localization. The K + channel toxins are small basic proteins with 31– 41 residues and 3 or 4 disulfide bonds. Structural studies on K + channel toxins from scorpi- ons have revealed a conserved three-dimensional (3D) scaffold composed of an -helix connected by two disul- fide bonds to one of the strands in a -sheet in the DB motif (3). A third disulfide bond connects the -sheet to an extended section in the amino-terminal portion of the toxin. These conserved secondary struc- tural elements display considerable primary sequence variability (Table I) that affects the affinity of the tox- ins for the various K + channels. The K + channel toxins have been grouped into sub- families based upon sequence similarities (Table I). Charybdotoxin (ChTX) is the most studied of the K + channel toxins. Eight residues (* residues in Table I) of ChTX were found to be critical for binding to the K + channel. A phenylalanine at position 2 and a trypto- phan at position 14 characterize the ChTX family (sub- family 1). Noxiustoxin (NTX) was the first K + channel toxin isolated. The NTX family (subfamily 2) is char- 1 This study was supported in part by NSF Grant MCB-9630775, NCI Grant CA13148 (NMR and Mass Spectrometry Facilities), and the Biotechnology Program Fund of the ECU School of Medicine. The atomic coordinates (PDB codes 1C55 and 1C56) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://ww- w.rcsb.org/). The chemical shifts were deposited in the BioMagRes- Bank (Accession Code: 4443). 2 To whom correspondence and reprint requests should be ad- dressed. Fax: (205) 934-6475. E-mail: nrkrishna@bmg.bhs.uab.edu. 18 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 379, No. 1, July 1, pp. 18 –27, 2000 doi:10.1006/abbi.2000.1858, available online at http://www.idealibrary.com on