Biochemistry Article Figure 1. Amino acid sequences and alignment of kurtoxin and five scorpion α-toxins. LqhαIT and Lqq III are highly active in insects. Lqh III and Bmk M1 are α-like toxins. AaH II is highly active in mammals. These sequences were aligned using ClustalX. Highly conserved residues are shaded in black or gray. The secondary structure elements of kurtoxin are shown as arrows (β-strand), bars (α-helix), and lines (connecting loops). terminal end of S3 and the N-terminal end of S4.17−19,38−42 The X-ray structures of voltage-gated K+ channels (i.e., KvAP and Kv1.2 channels) show that these regions of S3 and S4 form a helix−turn−helix motif termed the voltage sensor pad- dle.4,43,44 Gating modifier toxins that partition into the membrane interact with the voltage sensor paddle at the protein−lipid interface.42,45 Cav3 (T-type) voltage-gated Ca2+ channels can be differ- entiated from other types of Ca2+ channels on the basis of their activation at lower voltages, faster inactivation, slower deactivation, and smaller Ba2+ conductances.46 Their unique gating properties allow Cav3 (T-type) Ca2+ channels to trigger low-threshold spikes that can lead to burst firing and oscillatory behavior and can contribute to standing calcium currents near the resting membrane potential in a variety of cell types.47−50 Although these characteristics imply Cav3 (T-type) channels could play important roles in many tissues,51,52 progress in understanding their subunit composition and physiological functions has been hindered by a scarcity of ligands that interact with these channels.53,54 A gating modifier kurtoxin, isolated from the venom of the scorpion Parabuthus transvaalicus, is the first peptide ligand known to act on Cav3 (T-type) voltage-gated Ca2+ channels.55,56 Here, we describe the solution structure of kurtoxin determined using proton two-dimensional (2D) and heteronuclear three-dimensional (3D) NMR spectroscopy with dynamical simulated annealing calculations. The structure of kurtoxin closely resembles those of scorpion α-toxins (Figure 1) targeting Na+ channels and shows the unique structural characteristic of gating modifiers, electropositive and hydro- phobic patches on the surface of the molecule.39,57,58 Detailed inspection of the structure of kurtoxin offers the possibility of understanding the molecular basis of its Cav3 (T-type) Ca2+ channel selectivity and could facilitate clarification of the gating mechanism of voltage-gated ion channels. ■ EXPERIMENTAL PROCEDURES Sample Preparation. Functional kurtoxin was obtained using a bacterial expression system.59 The recombinant kurtoxin was expressed as an inclusion body, solubilized in a denaturing solution, and then refolded in a refolding solution. The crude folded kurtoxin was purified by preparative RP- HPLC, after which the purity of the recombinant protein was confirmed by analytical RP-HPLC and MALDI-TOF MS measurements. CD Spectral Analysis. CD spectra were recorded on a JASCO J-750 spectropolarimeter in a solution containing 0.01 M sodium phosphate in H2O and 0, 10, 15, 20, 25, or 30% CH3CN at pH 7.0. Measurements were taken at 20 °C using a quartz cell with a 1 mm path length. The spectra were expressed as molecular ellipticity [θ] in degrees square centimeter per decimole. NMR Spectroscopy. NMR measurements were taken on a Bruker AVANCE 600 spectrometer equipped with an xyz gradient triple-resonance probe. The samples used for proton 2D NMR experiments were 1 mM kurtoxin dissolved in water containing 25% CD3CN at pH 4.0 (uncorrected for the isotope effect). All proton 2D NMR spectra were recorded in a phase- sensitive mode using time-proportional phase incrementation (TPPI) for quadrature detection in the t1 dimension at 278, 288, and 298 K. TOCSY spectra were recorded using a MLEV- 17 pulse scheme60 with isotropic mixing times of 60 and 90 ms. NOESY spectra60−62 were recorded with mixing times of 60, 100, and 150 ms. Suppression of the solvent resonance in both the NOESY and TOCSY measurements was achieved using the WATERGATE scheme.63 E-COSY64 spectra were recorded to obtain the constraints for stereospecific assignments. The following triple-resonance 3D NMR spectra were recorded using 1 mM 13C- and 15N-labeled or 1 mM 15N- labeled kurtoxin in 25% CD3CN at 278 and 288 K. Uniformly 13C- and 15N-enriched kurtoxin was used to record 3D HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, and HN(CA)CO spectra.65−68 Uniformly 15N-enriched kurtox- in was used to record 2D 1H−15N HSQC69 and 3D 15N TOCSY-HSQC spectra with 90 ms mixing times and a 3D 15N NOESY-HSQC spectrum with a 120 ms mixing time. The 3JHN−Hα values were obtained from the 3D HNHA70 and 2D DQF-COSY spectra. Slowly exchanging backbone amide protons were identified by analysis of TOCSY spectra recorded in 75% D2O and 25% CD3CN on time scales of 30 min and 3.5 h and then every 3 h up to 25 h. 1H chemical shifts were referenced to DSS at 0 ppm, and 13C and 15N chemical shifts were calculated from the 1H frequency. All spectra were processed using AZARA version 2.5 (provided by W. Boucher) or XWIN-NMR and were analyzed using ANSIG version 3.371 on a Silicon Graphics Octane2 workstation or on a Linux workstation. NMR Experimental Restraints and Structure Calcu- α lations. The backbone NH−C H coupling constants were converted to backbone torsion angle ϕ constraints according to the following rules: for a 3JNH−CαH of <5.5 Hz, the ϕ angle was constrained in the range of −65 ± 25°; for a 3JNH−CαH of >8.0 Hz, it was constrained in the range of −120 ± 40°.72,73 Backbone dihedral constraints were not applied to 3JNH−CαH values between 5.5 and 8.0 Hz. The range of the χ1 side chain torsion angle constraints and the stereospecific assignment of the prochiral β-methylene protons were obtained using the 3Jαβ β coupling constants combined with the intraresidue NH−C H NOEs.74 The 3Jαβ coupling constants were determined from the E-COSY spectrum in D2O. For the t2g3, g2g3, and g2t3 conformations around the Cα−Cβ bonds, the χ1 side chain 1863 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873