Identification of a α-helical molten globule intermediate and structural characterization of β-cardiotoxin, an all β-sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

Abstract

β-Cardiotoxin is a novel member of the snake venom three-finger toxin (3FTX) family. This is the first exogenous protein to antagonize β-adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β-sheet peptides with 60-80 amino acid residues. Here, we describe the three-dimensional crystal structure of β-cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β-cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α-helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β-cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.

Keywords: beta-blocker; molten globule; non-hierarchical protein folding; thermal denaturation and structural transition; three-finger toxin.

Figure 1

Overall crystal structure and electron density omit map of β‐cardiotoxin. (a) Cartoon representation of the β‐cardiotoxin asymmetric unit (residues Ile43‐Cys55). Monomer A: cyan; monomer B: magenta; and monomer C: green. (b) Electron density map (2Fo‐Fc) showing the loop III region. The map is calculated by CCP4 program suit and contoured at 1σ. (c) Ribbon diagram showing the β‐cardiotoxin monomer (chain B). Disulfide bonds are presented as yellow sticks. N, C terminus and the three loops are labeled.

Figure 2

Comparison of β‐Cardiotoxin with other three‐finger toxins (3FTXs). (a) Sequence alignment of β‐cardiotoxin with all closely related 3FTX homologs (>90% identity). (b) Sequence alignment of β‐cardiotoxin with the conventional CTXs (about 50% identities). The alignment is done by ClustalW and figures are prepared by ESPRIPT. (c) Comparison of β‐cardiotoxin with its structural homologs using DALI search. β‐Cardiotoxin [3PLC] (chocolate), Hemachatoxin [3VTS] (60) (orange), cardiotoxin V [1KXI] (61) (red), cytotoxin 4 [4OM5] (62) (dark green), cytotoxin 3 [2BHI] (63) (light green), muscarinic m1‐toxin1 [2VLW] (64) (dark brown), cytotoxin 3 [1XT3] (65) (blue), cardiotoxin‐A3 [1H0J] (66) (light blue), bucain [2H8U] (67) (light brown), cytotoxin 2 [4OM4] (62) (yellow), muscarinic m1‐toxin1 [3NEQ] (68) (magenta), muscarinic toxin MT1 [4DO8] (68) (cyan), and ringhalexin (18) [4ZGY] (green). (d) Electrostatic surface potential of β‐cardiotoxin and Cardiotoxin V (61) (e). The electrostatic surface potentials in this paper are calculated using Pymol plug‐in APBS and scaled at ±10 kT. β‐Cardiotoxin does not have the cationic/hydrophobic central β‐sheet feature, which is critical for CTX's cytolytic activity.

Figure 3

Thermal (un)folding studies of β‐cardiotoxin. (a) Effect of temperature on the secondary structure of β‐cardiotoxin. The protein was dissolved in MilliQ water (0.5 mg/mL) and far‐UV CD spectra were recorded using a 0.1 cm path‐length cuvette 5°C (after cooling from 95°C, black dotted line). The blue and black arrows indicate the new bands arising at 203 and 222 nm. (b) Refolding of β‐cardiotoxin after thermal denaturation.

Figure 4

Effect of temperature on tertiary structure of β‐cardiotoxin. (a) β‐Cardiotoxin was dissolved in MilliQ water (1 mg/mL) and near‐UV CD spectra were recorded using a 0.1 cm path‐length cuvette. (b) Refolding of β‐cardiotoxin after thermal denaturation.

Figure 5

Chemical (un)folding studies of β‐cardiotoxin using guanidine hydrochloride. (a) Effect of guanidine HCl on secondary structure of β‐cardiotoxin. Chemical denaturation experiments were performed by dissolving β‐cardiotoxin in 25 mM Tris–HCL buffer pH 7.5 containing increasing concentrations of guanidine HCl. (b) Refolding of β‐cardiotoxin after the removal of guanidine HCl.

Figure 6

Chemical (un)folding studies of β‐cardiotoxin at different pH. (a) Effect of pH on secondary structure of β‐cardiotoxin. Chemical denaturation experiments were performed at 25°C by dissolving β‐cardiotoxin in buffers of different pH ranging from 2.5 to 9.5; 25 mM citrate pH 2.5, 25 mM MES pH 5.5, 25 mM MOPS pH 7.5, 25 mM Bis‐Tris propane pH 9.5.

Figure 7

Combined effect of temperature and pH on the secondary structure of β‐cardiotoxin. Thermal denaturation of β‐cardiotoxin at pH 1.5. The protein was dissolved in MilliQ water (0.5 mg/mL) and pH adjusted to 1.5 with HCl and far‐UV CD spectra (a) and near‐UV CD spectra (b) were recorded using a 0.1 cm path‐length cuvette. Refolding was observed at 5°C after cooling from 85°C, black dotted line. The blue and black arrows indicate the new bands arising at 203 and 222 nm.

Figure 8

Thermodynamic plots of (un)folding of β‐cardiotoxin. (a) Guanidine HCl‐induced (un)folding of β‐cardiotoxin. The line represents a fit to a two‐state model of (un)folding. (b) Van't Hoff analysis of the temperature‐induced (un)folding of β‐cardiotoxin. Open symbols refer to (un)folding in water, while closed symbols indicate denaturation of the protein at pH 1.5. The lines are the result of linear regression analyses.

Figure 9

Temperature dependent structural changes of β‐cardiotoxin by NMR. One‐dimensional proton NMR spectra of β‐ cardiotoxin showing upfield shifted aliphatic proton resonances (left panel) and downfield shifted amide proton resonances (right panel) at four different temperatures. The NMR spectra were acquired in an aqueous solution, pH 1.5.