Article pubs.acs.org/biochemistry Solution Structure of Kurtoxin: A Gating Modifier Selective for Cav3 Voltage-Gated Ca2+ Channels Chul Won Lee,†,‡ Chanhyung Bae,† Jaeho Lee,† Jae Ha Ryu,† Kenton J. Swartz,⊥ and Jae Il Kim*,† Ha Hyung Kim,§ Toshiyuki Kohno,∥ †Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea ‡Department of Chemistry, Chonnam National University, Gwangju 500-757, Republic of Korea §College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea ∥Department of Biochemistry, Kitasato University, School of Medicine, Kanagawa 252-0374, Japan ⊥Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, United States *S Supporting Information ABSTRACT: Kurtoxin is a 63-amino acid polypeptide isolated from the venom of the South African scorpion Parabuthus transvaalicus. It is the first and only peptide ligand known to interact with Cav3 (T-type) voltage-gated Ca2+ channels with high affinity and to modify the voltage-dependent gating of these channels. Here we describe the nuclear magnetic resonance (NMR) solution structure of kurtoxin determined using two- and three-dimensional NMR spectroscopy with dynamical simulated annealing calculations. The molecular structure of the toxin was highly similar to those of scorpion α-toxins and contained an α-helix, three β-strands, and several turns stabilized by four disulfide bonds. This so-called “cysteine-stabilized α-helix and β-sheet (CSαβ)” motif is found in a number of functionally varied small proteins. A detailed comparison of the backbone structure of kurtoxin with those of the scorpion α-toxins revealed that three regions [first long loop (Asp8−Ile15), β-hairpin loop (Gly39−Leu42), and C-terminal segment (Arg57−Ala63)] in kurtoxin significantly differ from the corresponding regions in scorpion α-toxins, suggesting that these regions may be important for interacting with Cav3 (T-type) Ca2+ channels. In addition, the surface profile of kurtoxin shows a larger and more focused electropositive patch along with a larger hydrophobic surface compared to those seen on scorpion α-toxins. These distinct surface properties of kurtoxin could explain its binding to Cav3 (T-type) voltage-gated Ca2+ channels. V and play a crucial role in regulating membrane potential oltage-gated ion channels are expressed by nearly all cells and a variety of cellular functions. These channels are comprised of two principle domains: a central pore domain formed by two segments, S5 and S6, and four surrounding voltage-sensing domains, each composed of segments S1− S4.1−4 Venomous animals (spiders, scorpions, and cone snails, among others) produce a broad array of polypeptide toxins, many of which bind to voltage-gated Na+, K+, or Ca2+ channels,5−8 and have proven to be valuable pharmacological tools for evaluating specific channel characteristics. Although the origins of the venomous peptide toxins that interact with voltage-gated ion channels are diverse, their modes of action fall into two major categories, pore blockade and gating modification, based on the domain with which they interact and their mechanisms of action. Pore blockers bind to the external vestibule of the channel and physically obstruct the movement of ions by occluding the ion-conducting pore.9 The three-dimensional structures of many pore-blocking toxins, including the μ-conotoxins for the Na+ channel, charybdotoxin for the K+ channel, and ω-conotoxins for the Ca2+ channel, have all been determined, allowing investigation of the structure−function relationships of the pore-forming domains of the channels.10−15 Gating modifiers, on the other hand, bind to the voltage-sensing domains of voltage-gated ion channels and modify the energetics of either activation or inactiva- tion.16−21 Established gating modifiers include the α- and β- scorpion toxins, sea anemone toxins, and δ-conotoxins for Na+ channels;22−28 hanatoxin (HaTx), SGTx1, GxTx-1E, and VSTx for K+ channels;29−33 and ω-agatoxin IVA (ω-Aga IVA) and ω- grammotoxin SIA (GrTx) for Ca2+ channels.34−37 Studies of the structure and function of gating modifiers have advanced our understanding of the molecular structures and gating mechanisms of voltage-gated ion channels. Studies employing HaTx, SGTx1, GxTx-1E, VSTx, GrTx, and ω-Aga IVA have revealed that, within voltage-gated K+ and Ca2+ channels, these proteins bind to structurally conserved motifs composed of hydrophobic and acidic residues within the C- Received: October 27, 2011 Revised: February 13, 2012 Published: February 13, 2012 © 2012 American Chemical Society 1862 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873