Biochemistry Kurtoxin contains three β-strands comprised of residues Ile2− Gly4, Ser34−Cys37, and Cys44−Leu49, which are arranged in an antiparallel fashion with several turns (Figure S3 of the Supporting Information). Our criteria allowed discrimination of the peripheral and central strands within the β-sheet. Structure Calculations. The structure of kurtoxin was determined from a total of 894 NMR experimental constraints, including 856 experimental distance constraints and 38 dihedral angle constraints, which correspond to an average of 14.2 constraints per residue. Of the 861 distance constraints, there were 311 intraresidue and 492 interresidue NOE distance constraints, 42 hydrogen bond constraints determined from hydrogen−deuterium exchange-out experiments, and 12 disulfide bond constraints. The 42 distance constraints related to hydrogen bonds were as follows: I2(HN)−L49(CO), G4(HN)−C46(CO), D25(HN)−K21(CO), C27(HN)− C23(CO), K28(HN)−N24(CO), G29(HN)−L26(CO), L30(HN)−C27(CO), K31(HN)−K28(CO), A32(HN)− C27(CO), G35(HN)−D33(CO), Y36(HN)−Y45(CO), W38(HN)−S43(CO), C44(HN)−I15(CO), Y45(HN)− Y36(CO), C46(HN)−G4(CO), Q47(HN)−S34(CO), L49(HN)−I2(CO), A53(HN)−P50(CO), K56(HN)− Y5(CO), R62(HN)−W10(CO), and A63(HN)−C61(CO). The disulfide bond pattern of kurtoxin was determined to be Cys12−Cys61, Cys16−Cys37, Cys23−Cys44, and Cys27−Cys46, based on sequential cleavage with proteases and MALDI-TOF MS measurements.59 We conducted the simulated annealing calculations starting with 100 random kurtoxin structures. From those, we selected 20 final structures (Figure 4A) that were in good agreement with the NMR experimental constraints (NOE distance and torsion angle violations of <0.2 Å and <2°, respectively). Statistics for the converged structures were evaluated in terms of the structural parameters (Table 1). The deviations from the idealized covalent geometry were very small, and the Lennard- Jones van der Waals energy was large and negative (−212.55 ± 19.40), indicating there were no distortions or nonbonded bad contacts in the converged structures. The atomic rmsd about the mean coordinate positions was 0.87 ± 0.14 Å for the backbone atoms (N, Cα, and C) and 1.54 ± 0.19 Å for all heavy atoms. Ramachandran analysis showed that 98.7% of all residues fell within allowed regions. Structure Description. The molecular structure of kurtoxin has a compact core consisting of an α-helix and a triple-stranded antiparallel β-sheet stabilized by four disulfide bridges. The 2.5-turn α-helix is composed of residues extending from Lys21 to Leu30 and is linked to the central strand of the β- sheet by two disulfide bridges (Cys23−Cys44 and Cys27−Cys46) (Figure 4B). The three β-strands are formed by residues Ile2− Gly4 (β-strand I), Ser34−Cys37 (β-strand II), and Cys44−Leu49 (β-strand III), with residues Ile2, Gly48, and Leu49 involved in a β-bulge conformation. The last two residues of β-strand III (Gly48 and Leu49) form a classical β-bulge and interact with the first residue of β-strand I (Ile2). As a result, the ϕ angle of Gly48 is positive (105.6°), which causes a distortion in the β-sheet. Two long loops (loop I, Tyr5−Asn20; loop II, Pro50−Ala63) extend from the core (Figure 4B). Long loop I includes two type IV β-turns (Asp8−Asn11 and Asn11−Arg14) and adopts the positive ϕ angles (58.9° and 89.9°, respectively) of Asn11 and Arg14 in the average structure. Long loop I is connected to β- strand II by a disulfide bond (Cys16−Cys37) and to the C- terminus by a disulfide bond (Cys12−Cys61) and a hydrogen bond [R62(HN)−W10(CO)]. Long loop II starts with a well- Article Table 1. Structural Statistics for the 20 Lowest-Energy Structures of Kurtoxina rmsd from experimental distance constraints (Å)b 0.0115 ± 0.0012 (856) rmsd fom experimental dihedral constraints (deg)b 0.1555 ± 0.0012 (38) energetic statistics (kcal/mol)c FNOE 5.7261 ± 1.2582 Ftor 0.9845 ± 0.0964 Frepel 5.3839 ± 1.3992 EL‑J −212.55 ± 19.4000 rmsd from idealized geometry bonds (Å) 0.0017 ± 0.0001 angles (deg) 0.5010 ± 0.0065 impropers (deg) 0.3391 ± 0.0060 Ramachandran analysisd (%) most favored regions 56.0 additionally allowed regions 36.1 generously allowed regions 6.5 disallowed regions 1.3 average rmsd (Å) backbone (N, Cα, C) 0.87 ± 0.14 all heavy atoms 1.54 ± 0.19 MolProbity analysise Clash score 94.39 ± 9.94 MolProbity score 4.37 ± 0.08 aNone of these 20 structures exhibited distance violations of >0.2 Å or dihedral angle violations of >2°. bThe number of each experimental constraint used in the calculations is given in parentheses. cFNOE, Ftor, and Frepel are the energies related to the NOE violations, the torsion angle violations, and the van der Waals repulsion term, respectively. The values of the force constants used for these terms are the standard values as depicted in the X-PLOR 3.1 manual. EL‑J is the Lennard- Jones van der Waals energy calculated with the CHARMm empirical energy function.102 EL‑J was not used in the dynamical simulated annealing calculations. dPROCHECK_NMR was used to assess the stereochemical quality of the structures. eThe MolProbity webserver was used to evaluate the determined kurtoxin ensemble structures. defined type I β-turn structure (residues 50−53) and extends to the C-terminus. It is stabilized through hydrogen bonding [ N 1 1 ( H D 2 1 ) − S 5 8 ( C O ) , K 5 6 ( H N ) − Y 5 ( C O ) , a n d R62(HN)−W10(CO)] and formation of a disulfide bridge (Cys12−Cys61) to long loop I. The short loop regions (Lys13− Ile15 and Trp40−Thr41) and the six C-terminal residues (Ser58− Ala63) are less defined in the final 20 structures than the other regions in kurtoxin (Figure 4C). This may reflect a lack of medium- and long-range NOE constraints due to the inherent flexibility of these regions. Two surface hydrophobic patches were observed in the kurtoxin structure. The major patch is composed of the solvent- exposed side chains of Tyr5, Tyr36, Trp38, Trp40, Leu42, and Tyr45 (Figure 4D), which, except for Tyr5, are located in the hairpin structure (strands II and III). Their side chains are well stacked, creating a compact hydrophobic patch on the surface of the molecule. The minor hydrophobic patch is situated on the opposite side of the protein and is centered around Trp17, Tyr18, Tyr22, and Leu26 (Figure 4D). Interestingly, all line- broadened residues (including Tyr17, Tyr18, Trp38, Gly39, Trp40, and Leu42) in pure water are located within either the major or minor surface hydrophobic patch in kurtoxin, strongly suggesting that the aggregation of kurtoxin results from these hydrophobic surface properties of kurtoxin. All of the charged residues except Asp8 are highly exposed on the surface of 1866 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873